Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions

  1. Kento Morimoto
  2. Hayate Suzuki
  3. Akihiro Kuno
  4. Yoko Daitoku
  5. Yoko Tanimoto
  6. Kanako Kato
  7. Kazuya Murata
  8. Fumihiro Sugiyama
  9. Seiya Mizuno

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Abstract

Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. In contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N -ethyl- N -nitrosourea (ENU) and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named C RISPR-& T ransposase-based R egiona L Mutagenesis (CTRL-Mutagenesis). We demonstrated that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.

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    Reply to the reviewers

    *Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    *Major comments:

    1. Mirc56_2 and 4 showed lower integration rates, and the authors suggest that this could be due to sgRNA pool imbalance. The authors should validate this by performing sequencing of the input sgRNA and cassettes. *

    →Thank you very much for your comment, and we agree with your suggestion.

    We are going to confirm sgRNA pool imbalance in donor vector library by amplicon short-read NGS.

    In addition, to confirm another possibility that we raised, we re-sequenced sgRNA donor vector for Mirc56_2 and 4, and will add the following sentences:

    “We firstly doubted that their low integration frequencies were caused by any mutations on PB transposon of sgRNA donor vector, on especially ITR or ID that are important for integration efficiency [PMID: 15663772]. Therefore, we sequenced PB transposons for *Mirc56_2 *and 4 again. However, we could not find any mutations on their PB transposon.” following to “…efficiency or cell growth.” in the Discussion (page14, line 346)

    *2. Clonal analysis in Figure 5c is unclear a. Figure 5c indicates that all changes were homozygous (e.g. both alleles were deleted). Was this the case in all clones? Or were some mutations heterozygous? *

    →Thank you very much for your comment.

    We apologize for the misleading context.

    We targeted mono allele on X chromosome in male mES cells so that all mutations should be hemizygous as mentioned in the Result (page11, line 259-260)

    To enhance our study is monoallelic assessment, we will add the following sentence:

    “This study targeted mono allele on X chromosome in male mES cells so that all genotype on Mirc56 should be hemizygous and these mutations induced might be cis-mutation.” following to“…owing to six tandem repeats [37]” in the Result (page12, line 302)

    *b. Many clones in Figure 5c show that the entire region was deleted (all black dots). Could this be due to some experimental error or misinterpretation of the sequencing data, or could it be validated using some orthogonal method? This is especially surprising for clones in which the final guide (Mirc56_13) was not detected yet the final site (Mirc56_13) was reported as "Regional deletion". *

    →Thank you very much for your comment.

    We apologize for the misleading context.

    Firstly, we just confirmed and sequenced the mature-miRNA genomic regions by amplifying approximately 200 bp around the target sites. Therefore, we defined unamplified regions as “miRNA deletion”. In addition, to make the Figure 5C easy to understand, we added “predicted regional deletion” and each name of clones as attached.

    In fact, only 4 clones harbored entire* Mirc56_X* deletions on all analysed Mirc56_X genomic region (Mirc56_1 to 13). Besides, these clones could be PCR-amplified by sgRNA cassettes and *Sry *on Y chromosome so that these results suggested we could successfully obtain their genomic DNA and at least mature-miRNA genomic regions were deleted.

    Moreover, Mirc56_13 deletions without target sites on Mirc56_13 are always within predicted regional deletions that are induced from upstream and downstream of sgRNA target sites. Therefore, it could be estimated that these deletions were induced from the target sites on Mirc56_14, 15, 16, or 17 and upstream of Mirc56_13.

    To clarify them, we will add the following sentences:

    • “Four clones (#2_066, #1_021, #1_029 and #1_046) harboured entire Mirc56_X deletions on all analysed Mirc56_X genomic region. In addition to these clones, only 3 pairs (#2_019 and #2_084, #2_038 and #1_023, #1_016 and #1_027) harboured same combination of mutations.” following to “…combinations of mutations (Figure 5C).” in the Result (page16, line 378-380)
    • “Meanwhile, focusing on relationship between mutations and target sites that targeted by sgRNA cassettes in each clone, all Mirc56_X genomic regions harbouring Indel mutations were target Micr56_X In addition, if sequential Mirc56_Xs on the genome were deleted, the most upstream and downstream of Mirc56_Xs deleted were always on the target Mirc56_X sites except for #2_025 and #1_41.” following to “…combinations of mutations (Figure 5C).” in the Result (page12, line 304)
    • “Genotyping PCR amplified approximately 200 bp around the mature-miRNA genomic region. Unamplified region is defined as miRNA deletion (Black circle) and amplified region was determined as Indel mutation (Gray circle) or Intact by short-NGS. If sequential Mirc56_Xs on the genome were deleted, black translucent square indicates predicted regional deletion assumed that the genomic region flanked by miRNA deletions was also deleted. Besides, if miRNA deletion was induced in Mirc56_13 and the clone have target* Mirc56_X* on Mirc56_14, 15, 16, or 17” following to “…in each PB mES clone.” in the Figure legend (page23, line 575) Moreover, because we defined “miRNA deletion”, we will change ”regional deletion” to “miRNA deletion” where I mean “deletion of the mature-miRNA genomic regions” in the Result (page13, line 312) and the Discussion (page14, line 363)

    *3. Next-generation targeted sequencing of clones should be made publicly accessible. *

    →Thank you very much for your comment. We apologize for the inconvenience.

    We already informed Review commons that we made publicly available.

    We already described BioProject ID PRJNA996747 in the Data Availability (page16, line 383-384)

    4. OPTIONAL - Cassette integration number is understudied. One important aspect of tiling mutagenesis is the control over how many guides are present in each cell. The authors report an average of 4.7 cassettes/cell. This could be modulated by the amount of donor vector added, and indeed the authors performed titration experiments, but only with a fluorescent reporter readout. It would be very useful to know how the concentration of donor vector corresponds to the number of cassettes/cell - perhaps genotyping of clones from one or two additional experiments would be sufficient.

    → Thank you very much for your suggestion.

    We agree that cassette integration number is one important aspect of tiling mutagenesis.

    To investigate how many copies our concentration of donor vector could integrate, we are going to check actual copy numbers in several clones by qPCR.

    We think that it is other research to confirm “how the concentration of donor vector corresponds to the number of cassettes/cell”. The correlation might not be liner due to transposase overproduction inhibition (OPI) so that it would require huge amounts of experiments to confirm it. Our research is how CTRL-Mutations induce diverse mutations but not how property PB system have.

    *Minor comments:

    1. The background fails to acknowledge the work of CRISPR-Cas tiling screens (e.g. https://doi.org/10.1038/nbt.3450) or CRISPR-Cas in creating mutagenesis in cell lines (e.g. https://doi.org/10.1007/978-1-0716-0247-8_29) *

    → Thank you very much for your suggestion, and we agree with your suggestion.

    We will add to acknowledge previous studies for CRISPR-Cas tilling screens.

    • “Recently, targeted mutagenesis combined forward genetics and reverse genetics has been developed such as saturating mutagenesis and tiling mutagenesis that induce random mutation within target gene(s) [PMID: 25141179, 31586052, 27260157, 28118392]. This targeted mutagenesis can construct a mutant library harbouring subtly different mutations within a target gene(s) so that comparative analysis through the mutant library can screen out critical mutation(s) for biological processes. These random mutagenesises have also revealed the function of numerous coding genes” following to “…list of coding genes [6–8].” in the Introduction (page3, line 55-56)
    • “In addition, the saturating mutagenesis are limited in the length of target region due to an approach basing homology-directed repair although it could introduce random mutations on donor template library harbouring any combination and variety of mutations [PMID: 25141179]. On the other hand, the tiling mutagenesis could expand target length in principle because the length depends on multiplex guide RNA (gRNA) designed to target genomic region. Therefore, tiling mutagenesis has been employed to identify critical regions embedded in cis-elements [PMID: 26375006, 30612741, 26751173, 27708057, 28416141, 31784727]. Tiling mutagenesis requires editor such as Cas9 or epigenetic modifier fused to catalytically dead Cas9 (e.g. KRAB-dCas9), and a library containing multiplex gRNA tiling across target genomic region. In general, random single guide RNA (sgRNA) expression cassette are integrated into chromosomes of host cells by retrotransposon system.” following to “…within a narrow region.” in the Introduction (page4, line 82) However, we do not agree that we have to acknowledge previous report about KI or KO by single or double cut in cell lines (as you suggested that https://doi.org/10.1007/978-1-0716-0247-8_29) because it is obvious knowledge. Therefore, we will not add this paper.

    2. Figure 1 left 'ROI random mutant PB mES cell' should be horizontally aligned so Mir_1, Mir_2 and MirX align with the upper figure.

    → Thank you very much for your kind comment, and we agree with your suggestion.

    Therefore, we changed it in the Figure 1.

    *3. It is interesting and unexpected that some guides never induce indels, even in the absence of a regional deletion (e.g. Mirc56_3, Mirc56_7). Why might this be? Was there perhaps an error in the assignment of these guides to these cells? *

    → Thank you very much for your comment.

    As you mentioned, Mirc56_3, 4 and 7 had no indel. We appreciate that we can correct our mistakes by your suggestion. We corrected Figure 5D as attached. In addition, we will correct average Mirc56_X site as 22.6 from 22.7.

    These sgRNA also induced miRNA deletion with low frequency (Mirc56_3: 38.9%, Mirc56_4: 25.0% and Mirc56_7: 68.0%, Figure 5D). Moreover, every deleted Mirc56_3, 4 and *7 *was within predicted regional deletion except for target Mirc56_3 of PB mES clone #2_080 (revised Figure 5C).

    Therefore, we raised why some guides never induce indels even in the absence of a regional deletion, as “In addition to low frequencies, Indel mutation might disappear due to regional deletion if these sgRNAs could induce Indel mutation”.

    To clarify them, we will add the following sentences:

    • “In particularly, middle target sites such as Mirc56_3, 4 and 7 were induced only miRNA deletion or Intact (Figure 5D)” following to “…in our mutant library (Figure 5C, D).” in the Discussion (page14, line 364)
    • “In fact, every deleted Mirc56_3, 4 and 7 was within predicted regional deletion except for target Mirc56_3 of PB mES clone #2_080 (Figure 5C). In addition, these sgRNA induced mutation with low frequency (Mirc56_3: 38.9%, Mirc56_4: 25.0% and Mirc56_7: 68.0%, Figure S6). Therefore, we suspected that regional deletion and their low mutation introduction rate facilitated to disappear Indel mutation.” following to “…induced at target sites.” in the Discussion (page14, line 366)

    *4. Regarding Mirc56_2 and 4 integration, on line 34 the authors suggest that "We suspect this was caused by a technical error, such as an unequal amount of sgRNA donor vector or the sequence in sgRNA cassettes affecting integration efficiency or cell growth." sgRNA library imbalance would be a technical error, but integration affecting cell growth is not a technical error. This sentence should be reworded. *

    → Thank you very much for your comment.

    We apologize for the misleading sentence even though this paper was already English-reviewed by English language editor.

    We will reword that “We suspect this was caused by the sequence in sgRNA cassettes affecting integration efficiency or cell growth, or a technical error such as an unequal amount of sgRNA donor vector.” following to “…PB mES clones via FACS..” in the Discussion (page14, line 344)

    *5. Line 540 "ration" is the incorrect word - perhaps "ratio"? *

    → Thank you very much for your kind comment, and we are sorry for the typo.

    We will correct it in the Figure legend (page22, line 540).

    6. Plot 5b should be shown as a histogram rather than a swarm plot to show how many clones were in each category.

    → Thank you very much for your suggestion.

    In Figure 5B, we aimed to indicate the number of sgRNA cassette varieties in each clone but not distribution of the number of integrated sgRNA cassettes. Distribution of the number of integrated sgRNA cassettes in clone library matched with the frequency of target sites in Figure 5D.

    We already described the distribution data as “In addition, an average of 22.7* Mirc56_X* sites … the same frequency except for the Mirc56_2- and* 4-targeting cassettes.*” in the Result (page13, line 312-315)

    *Reviewer #1 (Significance (Required)):

    1. General assessment: The authors are successful in creating clonal cell lines bearing a variety of mutations. Unfortunately, the cell lines also have transposase-mediated insertion events of the sgRNA cassettes at unknown positions in the genome, which will hamper the interpretability of any experiment using these cell lines. The authors fail to justify the use of the transposase and integration of the sgRNA, especially compared to lentiviral transfection or RNPs which would produce edits at the region of interest. Alternately, integrated sgRNA cassettes could have been excised with Flp recombinase as in https://doi.org/10.1007/978-1-0716-0247-8_29. *

    → Thank you very much for your suggestion.

    We agree that we did not mention why we choose PiggyBac system compared to lentiviral delivery.

    Therefore, we will add the following sentences:

    • “In general, random single guide RNA (sgRNA) expression cassette are integrated into chromosomes of host cells by retrotransposon system. To identify combinations of critical region embedded in target regions, it would require diverse combinations of mutations or inactivation sites. To induce multiple mutations or inactivated sites, it requires multiple sgRNA cassettes integration. However, multiple integration of sgRNA cassettes have higher risk of non-targeted endogenous gene disruptions and may impair functional analysis [PMID: 23435812].” in the Introduction.
    • “Here, we proposed that DNA transposon system rather than retrotransposon system is more suitable to remove sgRNA cassettes from a mutant library. Transposons are genetic elements that can relocate between genomic sites and there are two types of transposons: (1) DNA transposon is transferred by a "cut and paste" mechanism in which the transposon sequence is cut directly from the genome, and (2) retrotransposon is transferred by a "copy and paste" mechanism in which the transposon sequence is transcribed into RNA and then integrated by reverse transcribed [PMID: 21958341]. Therefore, retrotransposon is never removed from the genome. DNA transposon such as PiggyBac, Sleeping Beauty and Tol2 systems are also used as gene transfer tools in vertebrates [PMID: 26481584]. Especially, PiggyBac leaves no footprint on integrated sites after transposons relocated while other DNA transposon system leaves small insertion on integrated sites [PMID: 34064900]. In addition, excision-only-PiggyBac transposase that can remove transposons but not integrate them, is developed [PMID: 27929521]. Only PiggyBac system can remove transposons carrying sgRNA cassettes from mutant library with no footprint. Therefore, we aimed to validate PiggyBac system for CRISPRko tilling mutagenesis.” in the Introduction. However, we are not going to mention comparison to RNPs because it is obvious that random sgRNA expressions is important key for random mutagenesis and design of random sgRNA treatments by RNP is difficult. The reason is that the target region might be cleaved by almost all sgRNA incorporated into cells. On the other hand, it is easier to design the number of sgRNA expression variety using the delivery system via integration into the chromosome because only integrate sgRNA are expressed.

    In addition, we could not agree that “integrated sgRNA cassettes could have been excised with Flp recombinase as in https://doi.org/10.1007/978-1-0716-0247-8_29.”

    This paper reports the concept that one EM7>neoR expression cassette flanked by Frt within KI allele could select intended-KI clone and then the cassette could remove by Flp recombinase. However, this approach is not suitable for our method because it causes structural mutation by recombination of multi Frt cassettes that are integrated into nearby genomic regions. Therefore, we will not mention it.

    *2. Additionally, the genotyping analysis is unclear, and seems to indicate that each clone bears homozygous mutations, with several clones showing deletions of the entire region. *

    → Thank you very much for your suggestion.

    We will revise them in Reviewer #1 Major comment 2a and b.

    *3. Advance: The authors are motivated to create clones using tiling mutagenesis. Tiling mutagenesis has already been performed without transposases (e.g. https://doi.org/10.1038/nbt.3450, https://doi.org/10.1371/journal.pone.0170445, https://doi.org/10.1038/s41467-019-12489-8) in the context of a screen, and clones have already been created using CRISPR/Cas9 mutagenesis so the advance presented in this manuscript over previous published work is unclear. *

    →Thank you very much for your suggestion, and we agree with your suggestion.

    We will add to acknowledge previous studies for CRISPR-Cas tilling screens.

    We will add the following sentences:

    • “Recently, targeted mutagenesis combined forward genetics and reverse genetics has been developed such as saturating mutagenesis and tiling mutagenesis that induce random mutation within target gene(s) [PMID: 25141179, 31586052, 27260157, 28118392]. This targeted mutagenesis can construct a mutant library harbouring subtly different mutations within a target gene(s) so that comparative analysis through the mutant library can screen out critical mutation(s) for biological processes. These random mutagenesises have also revealed the function of numerous coding genes” following to “…list of coding genes [6–8].” in the Introduction (page3, line 55-56)
    • “In addition, the saturating mutagenesis are limited in the length of target region due to an approach basing homology-directed repair although it could introduce random mutations on donor template library harbouring any combination and variety of mutations [PMID: 25141179]. On the other hand, the tiling mutagenesis could expand target length in principle because the length depends on multiplex guide RNA (gRNA) designed to target genomic region. Therefore, tiling mutagenesis has been employed to identify critical regions embedded in cis-elements [PMID: 26375006, 30612741, 26751173, 27708057, 28416141, 31784727]. Tiling mutagenesis requires editor such as Cas9 or epigenetic modifier fused to catalytically dead Cas9 (e.g. KRAB-dCas9), and a library containing multiplex gRNA tiling across target genomic region. In general, random single guide RNA (sgRNA) expression cassette are integrated into chromosomes of host cells by retrotransposon system.” following to “…within a narrow region.” in the Introduction (page4, line 82) The paper you raised as DOI: https://doi.org/10.1038/nbt.3450 applied CRISPRko tiling mutagenesis to find out critical region embedded 2 kb of p53 binding enhancer region by lentiviral delivery of sgRNA cassettes. Our method employs PiggyBac system that can remove the sgRNA cassettes from the chromosome with no footprint. Therefore, our method should be novel method that generates mutant library with no risk of non-targeted endogenous gene disruptions. In addition, we expand the length of target region to more than 50 kb. This is one of the advances.

    The paper you raised as DOI: https://doi.org/10.1371/journal.pone.0170445 applied CRISPRko tiling mutagenesis to find out critical mutation on MAP2K1 and BRAF protein coding sequence by lentiviral delivery of sgRNA cassettes. Our method employs PiggyBac system that can remove the sgRNA cassettes from the chromosome with no footprint. Therefore, our method should be novel method that generates mutant library with no risk of non-targeted endogenous gene disruptions.

    The paper you raised as DOI: https://doi.org/10.1038/s41467-019-12489-8 applied CRISPRko tiling mutagenesis for to find out critical domain from protein coding sequence by lentiviral delivery of sgRNA cassettes. Our method employs PiggyBac system that can remove the sgRNA cassettes from the chromosome with no footprint. Therefore, our method should be novel method that generates mutant library with no risk of non-targeted endogenous gene disruptions.

    To clarify the advantages, we will add the following sentences:

    • “Here, we proposed that DNA transposon system rather than retrotransposon system is more suitable to remove sgRNA cassettes from a mutant library. Transposons are genetic elements that can relocate between genomic sites and there are two types of transposons: (1) DNA transposon is transferred by a "cut and paste" mechanism in which the transposon sequence is cut directly from the genome, and (2) retrotransposon is transferred by a "copy and paste" mechanism in which the transposon sequence is transcribed into RNA and then integrated by reverse transcribed [PMID: 21958341]. Therefore, retrotransposon is never removed from the genome. DNA transposon such as PiggyBac, Sleeping Beauty and Tol2 systems are also used as gene transfer tools in vertebrates [PMID: 26481584]. Especially, PiggyBac leaves no footprint on integrated sites after transposons relocated while other DNA transposon system leaves small insertion on integrated sites [PMID: 34064900]. In addition, excision-only-PiggyBac transposase that can remove transposons but not integrate them, is developed [PMID: 27929521]. Only PiggyBac system can remove transposons carrying sgRNA cassettes from mutant library with no footprint. Therefore, we aimed to validate PiggyBac system for CRISPRko tilling mutagenesis.” in the Introduction.
    • “CRISPRko tiling mutagenesis is conducted for less than 15 kb target genomic region so far [PMID: 26375006, 30612741], while CRISPRi tiling mutagenesis can target more than 70 kb [PMID: 27708057], it is reported that. Hence, it remains unknown unclear how length CRISPRko tiling mutagenesis could expand” in the Introduction. In addition, we are going to conduct transposon removal by exicision-only-PBase treatment with several PB mES clones, for the proof of concept that CTRL-Mutagenesis can generate mutant library with no sgRNA cassettes.

    *4. Audience: The manuscript is written for the basic research audience, and the method could be applied to the study of regions of interest in many diseases. However, the unexcised use of transposases make the method less desirable than other methods. *

    → Thank you very much for your suggestion.

    We do not agree that the PiggyBac make the method less desirable than other methods.

    As mentioned in our response for reviewer #1 Significance 3, only PiggyBac system can remove transposons carrying sgRNA cassettes from mutant library with no footprint. Multiple integration of sgRNA cassettes have higher risk of non-targeted endogenous gene disruptions and may impair functional analysis. However, sgRNA cassettes by lentiviral delivery is never removed from the genome. In addition, other approaches such as Flp recombinase that reviewer #1 proposed in Significance 1 is not better than PiggyBac because Flp recombinase causes stratal mutation by recombination of multi Frt cassettes that are integrated into nearby genomic regions.

    To clarify them, we will add the following sentences:

    • “However, multiple integration of single guide RNA (sgRNA) cassettes has higher risk of non-targeted endogenous gene disruptions and may impair functional analysis.” in the Abstract.
    • “However, multiple integration of sgRNA cassettes have higher risk of non-targeted endogenous gene disruptions and may impair functional analysis [PMID: 23435812]. To eliminate the risk that integrated sgRNA cassettes disrupt non-targeted endogenous genes, it is best way to remove the sgRNA cassettes from the chromosome.” in the Introduction.
    • “Here, we proposed that DNA transposon system rather than retrotransposon system is more suitable to remove sgRNA cassettes from a mutant library. Transposons are genetic elements that can relocate between genomic sites and there are two types of transposons: (1) DNA transposon is transferred by a "cut and paste" mechanism in which the transposon sequence is cut directly from the genome, and (2) retrotransposon is transferred by a "copy and paste" mechanism in which the transposon sequence is transcribed into RNA and then integrated by reverse transcribed [PMID: 21958341]. Therefore, retrotransposon is never removed from the genome. DNA transposon such as PiggyBac, Sleeping Beauty and Tol2 systems are also used as gene transfer tools in vertebrates [PMID: 26481584]. Especially, PiggyBac leaves no footprint on integrated sites after transposons relocated while other DNA transposon system leaves small insertion on integrated sites [PMID: 34064900]. In addition, excision-only-PiggyBac transposase that can remove transposons but not integrate them, is developed [PMID: 27929521]. Only PiggyBac system can remove transposons carrying sgRNA cassettes from mutant library with no footprint. Therefore, we aimed to validate PiggyBac system for CRISPRko tilling mutagenesis.” in the Introduction. In addition, we are going to conduct transposon removal by exicision-only-PBase treatment with several PB mES clones, for the proof of concept that CTRL-Mutagenesis can generate mutant library with no sgRNA cassettes.

    *Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Major concerns:

    1. Concern about the Novelty of Functional Analysis Platforms: The authors claim that there are no established platforms for the study of cis-elements or microRNA clusters. This assertion seems inaccurate, as previous studies have utilized Cas9 tiling screens to investigate cis-regulatory elements (CREs) and large-scale screens to probe microRNA functions, as exemplified by the works of Canver et al. in Nature 2015, Gasperini et al. in Cell 2019, and others. *

    → Thank you very much for your suggestion, and we agree with your suggestion.

    We apologize our false claim so that we will delete the following sentences:

    • “In contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap” in the Abstract (page2, line 28-30)
    • “While loss-of-function analysis has been conducted for numerous coding genes, very limited progress has been made on non-coding genes and cis-elements.” in the Introduction (page3, line 47-49) The paper you raised as DOI: https://doi.org/10.1038/nature15521 (Canver et al. in Nature 2015) applied CRISPRko tiling mutagenesis to find out critical region embedded 12 kb of BCL11A enhancer region by lentiviral delivery of sgRNA cassettes. Our method employs PiggyBac system that can remove the sgRNA cassettes from the chromosome with no footprint. Therefore, our method should be novel method that generates mutant library with no risk of non-targeted endogenous gene disruptions. In addition, we expand the length of target region to more than 50 kb. This is one of the advances.

    The paper you raised as DOI: https://doi.org/10.1016/j.cell.2018.11.029 (Gasperini et al. in Cell 2019) applied CRISPRko tiling mutagenesis to find out critical region embedded maximum 12 kb enhancer candidates, in addition to CRISPRi tilling candidate screening through one sgRNA by one candidate enhancer, by lentiviral delivery of sgRNA cassettes. Our method employs PiggyBac system that can remove the sgRNA cassettes from the chromosome with no footprint. Therefore, our method should be novel method that generates mutant library with no risk of non-targeted endogenous gene disruptions. In addition, we expand the length of target region to more than 50 kb. This is one of the advances. Additionally, to identify combinations of critical region embedded in target regions, it would require diverse combinations of mutations or inactivation sites. To induce multiple mutations or inactivated sites, it requires multiple sgRNA cassettes integration. However, multiple integration of sgRNA cassettes have higher risk of non-targeted endogenous gene disruptions and may impair functional analysis. To eliminate the risk that integrated sgRNA cassettes disrupt non-targeted endogenous genes, it is best way to remove the sgRNA cassettes from the chromosome. Thus, to identify combinations of critical region embedded in target regions with no artifact owing to no footprint by removal of sgRNA cassettes, CRISPRko tiling mutagenesis rather than CRISPRi is better method because CRISPRi requires integrated cassettes that stably expressed sgRNA and epigenetic modifier fused to dCas9. Therefore, our method should be novel method that generates mutant library with no risk of non-targeted endogenous gene disruptions to find out combinations of critical region embedded in target regions.

    Therefore, to add to acknowledge previous studies and clarify the advantages, we will add the following sentences:

    • “In addition, the saturating mutagenesis are limited in the length of target region due to an approach basing homology-directed repair although it could introduce random mutations on donor template library harbouring any combination and variety of mutations [PMID: 25141179]. On the other hand, the tiling mutagenesis could expand target length in principle because the length depends on multiplex guide RNA (gRNA) designed to target genomic region. Therefore, tiling mutagenesis has been employed to identify critical regions embedded in cis-elements [PMID: 26375006, 30612741, 26751173, 27708057, 28416141, 31784727]. Tiling mutagenesis requires editor such as Cas9 or epigenetic modifier fused to catalytically dead Cas9 (e.g. KRAB-dCas9), and a library containing multiplex gRNA tiling across target genomic region. In general, random single guide RNA (sgRNA) expression cassette are integrated into chromosomes of host cells by retrotransposon system.” following to “…within a narrow region.” in the Introduction (page4, line 82)
    • “To identify combinations of critical region embedded in target regions, it would require diverse combinations of mutations or inactivation sites. To induce multiple mutations or inactivated sites, it requires multiple sgRNA cassettes integration. However, multiple integration of sgRNA cassettes have higher risk of non-targeted endogenous gene disruptions and may impair functional analysis [PMID: 23435812]. To eliminate the risk that integrated sgRNA cassettes disrupt non-targeted endogenous genes, it is best way to remove the sgRNA cassettes from the chromosome. Thus, to identify combinations of critical region embedded in target regions with no artifact owing to no footprint by removal of sgRNA cassettes, CRISPRko tiling mutagenesis rather than CRISPRi is better method because CRISPRi requires integrated cassettes that stably expressed sgRNA and epigenetic modifier fused to dCas9.” in the Introduction.
    • “Here, we proposed that DNA transposon system rather than retrotransposon system is more suitable to remove sgRNA cassettes from a mutant library. Transposons are genetic elements that can relocate between genomic sites and there are two types of transposons: (1) DNA transposon is transferred by a "cut and paste" mechanism in which the transposon sequence is cut directly from the genome, and (2) retrotransposon is transferred by a "copy and paste" mechanism in which the transposon sequence is transcribed into RNA and then integrated by reverse transcribed [PMID: 21958341]. Therefore, retrotransposon is never removed from the genome. DNA transposon such as PiggyBac, Sleeping Beauty and Tol2 systems are also used as gene transfer tools in vertebrates [PMID: 26481584]. Especially, PiggyBac leaves no footprint on integrated sites after transposons relocated while other DNA transposon system leaves small insertion on integrated sites [PMID: 34064900]. In addition, excision-only-PiggyBac transposase that can remove transposons but not integrate them, is developed [PMID: 27929521]. Only PiggyBac system can remove transposons carrying sgRNA cassettes from mutant library with no footprint. Therefore, we aimed to validate PiggyBac system for CRISPRko tilling mutagenesis.” in the Introduction.
    • “CRISPRko tiling mutagenesis is conducted for less than 15 kb target genomic region so far [PMID: 26375006, 30612741], while CRISPRi tiling mutagenesis can target more than 70 kb [PMID: 27708057], it is reported that. Hence, it remains unknown unclear how length CRISPRko tiling mutagenesis could expand” in the Introduction. On the other hand, we could not find previous studies employing Cas9 tiling mutagenesis to investigate miRNA functions. The application for miRNA cluster is also one of the advances.

    2) Advantages of PiggyBack System Over Lentiviral Integration: The paper does not clearly articulate the advantages of their proposed PiggyBack-based system for sgRNA integration over traditional lentiviral integration. Both methods facilitate the random integration of multiple gRNAs, but the paper lacks a comparative analysis or justification for choosing the PiggyBack system.

    → Thank you very much for your suggestion, and we agree with your suggestion.

    We agree that we did not mention why we choose PiggyBac system compared to lentiviral delivery.

    Therefore, we will add the following sentences:

    • “In general, random single guide RNA (sgRNA) expression cassette are integrated into chromosomes of host cells by retrotransposon system. To identify combinations of critical region embedded in target regions, it would require diverse combinations of mutations or inactivation sites. To induce multiple mutations or inactivated sites, it requires multiple sgRNA cassettes integration. However, multiple integration of sgRNA cassettes have higher risk of non-targeted endogenous gene disruptions and may impair functional analysis [PMID: 23435812].” in the Introduction.
    • “Here, we proposed that DNA transposon system rather than retrotransposon system is more suitable to remove sgRNA cassettes from a mutant library. Transposons are genetic elements that can relocate between genomic sites and there are two types of transposons: (1) DNA transposon is transferred by a "cut and paste" mechanism in which the transposon sequence is cut directly from the genome, and (2) retrotransposon is transferred by a "copy and paste" mechanism in which the transposon sequence is transcribed into RNA and then integrated by reverse transcribed [PMID: 21958341]. Therefore, retrotransposon is never removed from the genome. DNA transposon such as PiggyBac, Sleeping Beauty and Tol2 systems are also used as gene transfer tools in vertebrates [PMID: 26481584]. Especially, PiggyBac leaves no footprint on integrated sites after transposons relocated while other DNA transposon system leaves small insertion on integrated sites [PMID: 34064900]. In addition, excision-only-PiggyBac transposase that can remove transposons but not integrate them, is developed [PMID: 27929521]. Only PiggyBac system can remove transposons carrying sgRNA cassettes from mutant library with no footprint. Therefore, we aimed to validate PiggyBac system for CRISPRko tilling mutagenesis.” in the Introduction.

    *3) Lack of Comparative Analysis with Alternative Methods: The authors did not provide a comparison of CTRL-Mutagenesis with other existing screening methods. Such a comparison is crucial for understanding the effectiveness and efficiency of the new method in relation to established techniques. *

    → Thank you very much for your suggestion.

    We agree with the comparison is one of important experiments.

    However, our main claim is validation of tiling mutagenesis using PiggyBac that is only integration system with no footprint. Therefore, we propose our novelty without the comparison and not argue higher / lower efficiency of CTRL-Mutagenesis compared to exiting methods.

    *4) Limitations in Library Resolution: The paper acknowledges the limited resolution of their proposed library. The authors might have explored the use of base editors for enhanced resolution in such screens, as base editing could potentially offer more precise and controlled mutagenesis as briefly mentioned in the discussion. *

    → Thank you very much for your suggestion.

    We agree with your suggestion.

    Base editing is occurred within only editing window. In addition, a major limitation of prime editing is low efficiency (https://doi.org/10.1016/j.tibtech.2023.03.004). Therefore, design of sgRNA for base editor or pegRNA and its editing efficiency requires huge amounts of experiments.

    Our study is proof of concept to validate PiggyBac system for CRISPRko tilling mutagenesis and expanded the length of target regions. Thus, we just discussed limited resolution of our mutant library and proposed the use of base editors for enhanced resolution in the Discussion (page14, line 366-370).

    5) Absence of Functional Data Post-Mutagenesis: A significant limitation of the study is the absence of functional data following the creation of cells with different mutations. While the authors speculate about using differentiation systems or organoids for practical applications, they do not provide empirical data to demonstrate the utility of the CTRL-Mutagenesis approach. This lack of functional validation raises questions about the practical applicability of the method.

    → Thank you very much for your suggestion.

    We agree with your suggestion.

    We would make functional analysis future research.

    In this paper, we just validated PiggyBac system for CRISPRko tilling mutagenesis and expanded the length of target regions.

    To change our tone that claiming usability of our method for functional analysis, we will change the following sentences:

    • Change “to identify functionally important elements in non-coding regions” to “to induce diverse combination and variety of mutations within more than 50 kb non-coding region” in the Title.
    • Add “However, not much loss-of-function screens of non-coding regulatory elements has been conducted due to ambiguous annotations compared with protein-coding genes. Tiling mutagenesis has been employed to identify critical regions embedded in non-coding regulatory elements by comparative analysis through a mutant library harbouring subtly different regions mutated within less than 15 kb region. Conventional tiling mutagenesis construct a mutant library integrated multiple sgRNA cassettes by retroviral delivery. However, multiple integration of single guide RNA (sgRNA) cassettes has higher risk of non-targeted endogenous gene disruptions and may impair functional analysis. Herein, combining tiling mutagenesis and PiggyBac transposon that can be removed with no footprint on integrated sites, we established an expanded tilling mutagenesis method named CRISPR- & Transposase-based RegionaL Mutagenesis (CTRL-Mutagenesis). We demonstrated that PiggyBac system could integrated diverse combinations and varieties of sgRNA cassettes.and then CTRL-Mutagenesis randomly induces diverse combination and variety of mutations within more than 50 kb non-coding region in murine embryonic stem cells. CTRL-Mutagenesis would apply for wider non-coding regulatory elements with no risk of non-targeted endogenous gene disruptions.” in the Abstract.
    • Delete “Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.” in the Abstract (page2, line 38-40).
    • Change “The generated random mutant mES clone library could facilitate further functional analyses of non-coding regulatory elements within the genome.” to “The generated random mutant mES clone library could develop to investigate critical regions of non-coding regulatory elements within the genome.” In the Introduction (page4, line 88-90)

    *Reviewer #2 (Significance (Required)):

    1. In summary, while the idea to integrate sgRNA in the genome by the PiggyBack system is interesting the claim of novelty is questionable due to existing methods in the field. The advantages of their system over existing technologies are not clearly articulated, and a lack of comparative analysis with other methods leaves the efficiency of CTRL-Mutagenesis uncertain. *

    → Thank you very much for your suggestion.

    Previous studies about CRISPRko and CRISPRi tiling mutagenesis employ lentiviral delivery of sgRNA cassettes into the genome. However, multi sgRNA cassette integrations have higher risk to disrupt non-targeted endogenous functions. To eliminate the risk that integrated sgRNA cassettes disrupt non-targeted endogenous genes, it is best way to remove the sgRNA cassettes from the chromosome. Nevertheless, lentiviral transposon, one of retrotransposon, cannot be removed from the chromosome. On the other hand, only PiggyBac transposon can be removed with no footprint. Therefore, we aimed to validate PiggyBac system for tiling mutagenesis. Moreover, there is no report that CRISPRko tiling mutagenesis apply for more than 15 kb genomic region. Therefore, we aimed to expand the length of target region.

    Therefore, we will change our claim that our method could expand CRISPRko tiling mutagenesis to more than 50 kb with no risk of non-targeted endogenous gene disruption.

    We will add the novelty and advantage of our method.

    • “Here, we proposed that DNA transposon system rather than retrotransposon system is more suitable to remove sgRNA cassettes from a mutant library. Transposons are genetic elements that can relocate between genomic sites and there are two types of transposons: (1) DNA transposon is transferred by a "cut and paste" mechanism in which the transposon sequence is cut directly from the genome, and (2) retrotransposon is transferred by a "copy and paste" mechanism in which the transposon sequence is transcribed into RNA and then integrated by reverse transcribed [PMID: 21958341]. Therefore, retrotransposon is never removed from the genome. DNA transposon such as PiggyBac, Sleeping Beauty and Tol2 systems are also used as gene transfer tools in vertebrates [PMID: 26481584]. Especially, PiggyBac leaves no footprint on integrated sites after transposons relocated while other DNA transposon system leaves small insertion on integrated sites [PMID: 34064900]. In addition, excision-only-PiggyBac transposase that can remove transposons but not integrate them, is developed [PMID: 27929521]. Only PiggyBac system can remove transposons carrying sgRNA cassettes from mutant library with no footprint. Therefore, we aimed to validate PiggyBac system for CRISPRko tilling mutagenesis.” in the Introduction.
    • “CRISPRko tiling mutagenesis is conducted for less than 15 kb target genomic region so far [PMID: 26375006, 30612741], while CRISPRi tiling mutagenesis can target more than 70 kb [PMID: 27708057], it is reported that. Hence, it remains unknown unclear how length CRISPRko tiling mutagenesis could expand” in the Introduction. However, our main claim is validation of tiling mutagenesis using PiggyBac that is only integration system with no footprint. Therefore, we will propose our novelty without the comparison and not argue higher / lower efficiency of CTRL-Mutagenesis compared to exiting methods.

    In addition, we are going to conduct transposon removal by exicision-only-PBase treatment with several PB mES clones, for the proof of concept that CTRL-Mutagenesis can generate mutant library with no sgRNA cassettes.

    2. Moreover, the limited resolution of their library and the absence of functional data post-mutagenesis are significant drawbacks that need to be addressed in future research to ascertain the method's practical utility.

    → Thank you very much for your suggestion.

    We agree with your suggestion.

    We would make functional analysis future research.

    Base editing is occurred within only editing window. In addition, a major limitation of prime editing is low efficiency (https://doi.org/10.1016/j.tibtech.2023.03.004). Therefore, design of sgRNA for base editor or pegRNA and its editing efficiency requires huge amounts of experiments.

    Our study is proof of concept to validate PiggyBac system for CRISPRko tilling mutagenesis and expanded the length of target regions. Thus, we just discussed limited resolution of our mutant library and proposed the use of base editors for enhanced resolution in the Discussion (page14, line 366-370).

    Therefore, we just claimed that we validated PiggyBac system for CRISPRko tilling mutagenesis and expanded the length of target regions.

    *Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Major comments:

    1. Authors claim that "CTRL-mutagenesis randomly induces diverse mutations only within the targeted regions in murine embryonic stem (mES) cells.", however, the outcome of mutations is not entirely random since most of the mutations are regional deletions. For example, despite the random distribution of gRNAs per cell, the inner regions like Mirc56_5 or Mirc56_8 are mutated with >80% efficiency.*

    → Thank you very much for your comment.

    We agree that middle regions are tending to be deleted and mutation type induced is not entirely random. However, we do not agree that “the outcome of mutations is not entirely random since most of the mutations are regional deletions.” Focusing on the combinations of mutations as mentioned in the Result (page12, line 302-304), CTRL-Mutagenesis could induce diverse mutation combinations randomly at a moderate degree. In fact, 79.2% of clones harboring multiple mutations were induced different combinations of mutations. In addition, to confirm how mutations occurred within Mirc56 by CTRL-Mutagenesis, we constructed only 87 mutant clones though single cloning. Therefore, it is not completely understanded due to fewer clones compared with conventional CRISPRko tiling mutant library. Of course, we should improve the randomness of mutation combinations, but we already discussed it and proposed solutions in the Discussion (page14, line 366-370).

    Certainly, CTRL-Mutagenesis would be difficult to identify necessary and sufficient genomic region due to incomplete randomness. Nevertheless, there is no report to induce diverse combination and variety of mutations within more than 50 kb genomic region. Hence, CTRL-Mutagenesis should be worth screening out critical regions within more than 50 kb regions.

    To clarify them, will add the following sentences:

    • “CRISPRko tiling mutagenesis is conducted for less than 15 kb target genomic region so far [PMID: 26375006, 30612741], while CRISPRi tiling mutagenesis can target more than 70 kb [PMID: 27708057], it is reported that. Hence, it remains unknown unclear how length CRISPRko tiling mutagenesis could expand” in the Introduction.
    • “Four clones (#2_066, #1_021, #1_029 and #1_046) harboured entire Mirc56_X deletions on all analysed Mirc56_X genomic region. In addition to these clones, only 3 pairs (#2_019 and #2_084, #2_038 and #1_023, #1_016 and #1_027) were induced same combination of mutations. Besides, 26 clones had only one mutation from Mirc56_1 to Mirc56_13. On the other hand, there was no mutation on Mirc56_1 to 13 in 11 clones including 5 clones (#2_012, #2_015, #2_054, #2_092 and #2_102) carried no sgRNA cassette for Mirc56 _1 to 13 and 6 clones (#2_017, #2_053, #2_098, #1_003, #1_012 and #1_044) even carried any one of sgRNA cassettes for Mirc56 _1 to 13. Among 48 clones carrying multiple mutations except for clones carrying only one mutation or Intact, 38 clones (79.2%) harboured different combinations of mutations. These results suggested that CTRL-Mutagenesis could induce diverse combinations of mutations.” following to “…different combinations of mutations (Figure 5C).” in the Result (page12, line 304)
    • “Note that CTRL-Mutagenesis would be difficult to identify necessary and sufficient genomic region due to incomplete randomness. Nevertheless, CTRL-Mutagenesis should be worth screening out critical regions within more than 50 kb regions” following to “…to induce regional deletions.” in the Discussion (page15, line 378)
    • Change “diverse mutations” to “diverse combination and variety of mutations” in the Title, Abstract (page2, line 37), Introduction (page4, line 87), Result (page13, line 318), Discussion (page13, line 325), (page14, line 363) Additionally, we do not agree with your suggestions that “the inner regions like Mirc56_5 or Mirc56_8 are mutated with >80% efficiency”. We apologize for the misleading context. These high mutation rates were calculated on only the target sites. Actually, maximum mutation rate on all MIrc56_X genomic regions are 44.8% on Mirc56_10, minimum is 14.9% on Mirc56_2 and an average is 30.9% (attached Figure).

    We appreciate that we can recognize our misleading context by your suggestion. It is more important that the analysis focusing all Mirc56_X genomic regions rather than target Mirc56_X. Therefore, we newly made figure about event occurrence in Mirc56_X genomic regions (attached Figure) as Figure 5D and replaced previous Figure 5D about event occurrence in target Mirc56_X to Supplemental Figure S6.

    To clarify them, we will add the following sentences:

    • “As for event occurrences on each Mirc56_X genomic region, miRNA deletions were dominant and an average of 26.7 Mirc56_X genomic region were induced mutations in 87 clones (Figure 5D). Maximum mutation rate on all MIrc56_X genomic regions was 44.8% (39/87) on Mirc56_10, minimum was 14.9% (13/87) on Mirc56_2” following to “…on the same strand” in the Result (page12, line 309)
    • “__D, __Mutations in 87 Mirc56 random mutant clones. The target sites do not include* Mirc56_14, 15, 16*, and The vertical axis and bar graphs show event occurrence on each Mirc56 genomic region in 87 Mirc56 random mutant clones. The bar colour indicates each event (Black: Regional deletion, Gray: Indel mutation, White: Intact).” in the Figure legend.

    2. Also, although the authors discuss that the lower mutation frequency observed for Mirc56_2 and 4 may be due to a technical error, confirming this by repeating the experiment would be important to prove the usability of this method.

    →Thank you very much for your comment, and we agree with your suggestion.

    We had already constructed bulk PB mES cells twice and showed Figure 4B combined these experimental replicates.

    To clarify that we constructed bulk PB mES cells twice, we changed Figure 4B as attached and will add the following sentences:

    • “Even though these bulk PB mES cells were constructed twice, it seemed that sgRNA cassettes for *Mirc56_2 *and *4 *were difficult to integrate into the genome.” following to “…were rarely detected” in the Result (page11, line 273)
    • “In addition, we suspected technical errors so that we constructed bulk PB mES cells twice. Unfortunately, their low integration frequencies were not improved.” following to “…efficiency or cell growth.” in the Discussion (page14, line 346)
    • “Bulk1 and Bulk2 indicate the experimental replicate.” following to “…next-generation sequencing (NGS).”in the Figure legend (page22 line 563) In addition, we re-sequenced sgRNA donor vector for Mirc56_2 and 4, and will add the following sentences:

    “We firstly doubted that their low integration frequencies were caused by any mutations on PB transposon of sgRNA donor vector, on especially ITR or ID that are important for integration efficiency [PMID: 15663772]. Therefore, we sequenced PB transposons for *Mirc56_2 *and 4 again. However, we could not find any mutations on their PB transposon.” following to “…efficiency or cell growth.” in the Discussion (page14, line 346)

    Moreover, to confirm the technical error, we are going to confirm sgRNA pool imbalance in donor vector library by amplicon short-read NGS.

    *3. Additionally, the experiments were performed on the haploid X chromosome of a male cell line. It is questionable whether this method can be generalized to other regions located in the other chromosomes. Clarifying These points would be essential especially because the focus of this manuscript is to describe the efficiency of this novel methodology. *

    →Thank you very much for your comment.

    We expect that CTRL-Mutagenesis could be valid on other biallelic locus.

    Therefore, we raised predicted issue such as complex genotyping and proposed one solution.

    When we target other biallelic locus, we must determine whether the combination of mutations induced are cis- or trans-mutations. Haplotype phasing, combined long-read sequencing with SNP markers within ROI on maternal/paternal chromosome, assembles each allele via SNP markers on each read [PMID: 35710642]. Therefore, combining CTRL-Mutagenesis on heterozygotic alleles of cells derived from such as human or murine hybrid with haplotype phasing might simplify genotyping.

    We will add the following sentences:

    “In this study, CTRL-Mutagenesis was validated by genotyping on mono allele in male mES cells to avoid investigating whether the combination of mutations induced are cis- or trans-mutations. All genotypes on Mirc56 should be hemizygous and these mutations induced might be cis-mutations so that we determined the genotypes by amplifying approximately 200 bp around the target sites. However, we did not confirm large mutations such as deletion of the genomic region between target sites and inversion. Long-read sequencing might capture their large mutations. Besides, we also expect that CTRL-Mutagenesis could be valid for ROI on biallelic autosome and X chromosome in female. Therefore, it is required to determine whether the combination of mutations induced are cis- or trans-mutation. Haplotype phasing, combined long-read sequencing with SNP markers within ROI on maternal/paternal chromosome, assembles each allele via SNP markers on each read [PMID: 35710642]. Therefore, combining CTRL-Mutagenesis on heterozygotic alleles of cells derived from such as human or murine hybrid with haplotype phasing might simplify genotyping.” in the Discussion.

    *4. The limitations of the methods seem not to be fully described in the manuscript and must be clarified. Compared to the previous studies (see "significance" section for details), this method is inferior in that (1) it is time-consuming because it requires clonal expansion of single cells and (2) it has low throughput because it requires genome sequencing due to the occurrence of deletions. These points should be described for the potential users of this methodology. For example, it may be useful to detail the time consumption in each experimental step in Fig. 4A. *

    →Thank you very much for your comment.

    We do not agree that (1) it is time-consuming because it requires clonal expansion of single cells.

    To confirm the mutations that CTRL-Mutagenesis induced, we did not conduct phenotyping screening such as dropout screening in this study. For further high-throughput screening, CTRL-Mutagenesis could apply bulk mutant mES cells, that is treated with Cas9 and EGFP-positive, for phenotyping screening.

    Additionally, we do not agree that (2) it has low throughput because it requires genome sequencing due to the occurrence of deletions. In this study, to prove our concept that CTRL-Mutagenesis could induce diverse combinations and varieties of mutations such as Indel and regional deletion, we conducted genotyping in all random mutant clones. On the other hand, there are alternative comparative method to improve throughput without genotyping. Combination of phenotyping screening and gene expression assay for target miRNAs or transcript regulated by target cis-element help us obtain clones harboring mutations on functionally critical regions within target region. Finally, we should conduct genotyping to identify critical regions embedded in non-coding regulatory elements.

    Even so, we will add the time consumption in Figure 4A as attached because the information may be useful for potential users as you mentioned.

    *Minor comments:

    1. Data and methods are well-presented for reproducibility. The EGFP-positive ratio may be added to Fig. 4C for clarity. *

    →Thank you very much for your kind comment.

    We added the EGFP-positive ratio to Figure 4C and will add the following sentence:

    “The percentage above the box indicates the EGFP-positive ratio.” following to “…the gates of the EGFP filter.” in the Figure legends (page23, line 567)

    2. Enhance referencing accuracy, rectify DOI format in ref 21, and ensure consistency in citation formatting, e.g., ref 32.

    →Thank you very much for your kind comment.

    Along with the transfer, we will modify the style of references and have already confirmed the referencing accuracy in the Reference.

    3. It seems that the experimental condition (e.g. The amount of vectors used for transfection) should be re-considered every time the researcher wants to set up an experiment changing target genomic regions, cell types etc. If so, this also should be described in the text for potential users of this method.

    →Thank you very much for your comment, and we agree with your suggestion.

    We will add the following sentences:

    “This study just validated CTRL-Mutagenesis for 17 target sites in mES cells. Therefore, it might be better to adjust the number of integrated sgRNA cassettes according to the number of target sites and cell types.” following to “…sgRNA cassettes to be integrated.” in the Discussion (page14, line 355)

    *Reviewer #3 (Significance (Required)):

    There were various methods described in the late 2010's which aimed to screen for the functional non-coding regions using approaches such as KO-based, HDR-based, and epigenetic silencing using dCas9 (for example, PMID: 25141179, 26751173, 27708057, 28416141, 31784727). The authors should summarize what would be the strength of their method compared to these previously described methodologies. The strength of this methodology seems to be moderate complexity and cost-effectiveness compared to these previous techniques. It may be difficult for this methodology to become a state-of-the-art method to evaluate cis-element combinations, but it can be beneficial to researchers wanting to set up a low-cost system that can produce moderately complex cell libraries.*

    →Thank you very much for your suggestion.

    We will add to acknowledge previous studies for CRISPR-Cas tilling screens.

    • “Recently, targeted mutagenesis combined forward genetics and reverse genetics has been developed such as saturating mutagenesis and tiling mutagenesis that induce random mutation within target gene(s) [PMID: 25141179, 31586052, 27260157, 28118392]. This targeted mutagenesis can construct a mutant library harbouring subtly different mutations within a target gene(s) so that comparative analysis through the mutant library can screen out critical mutation(s) for biological processes. These random mutagenesises have also revealed the function of numerous coding genes” following to “…list of coding genes [6–8].” in the Introduction (page3, line 55-56)
    • “In addition, the saturating mutagenesis are limited in the length of target region due to an approach basing homology-directed repair although it could introduce random mutations on donor template library harbouring any combination and variety of mutations [PMID: 25141179]. On the other hand, the tiling mutagenesis could expand target length in principle because the length depends on multiplex guide RNA (gRNA) designed to target genomic region. Therefore, tiling mutagenesis has been employed to identify critical regions embedded in cis-elements [PMID: 26375006, 30612741, 26751173, 27708057, 28416141, 31784727]. Tiling mutagenesis requires editor such as Cas9 or epigenetic modifier fused to catalytically dead Cas9 (e.g. KRAB-dCas9), and a library containing multiplex gRNA tiling across target genomic region. In general, random single guide RNA (sgRNA) expression cassette are integrated into chromosomes of host cells by retrotransposon system.” following to “…within a narrow region.” in the Introduction (page4, line 82) The paper you raised as DOI: https://doi.org/10.1038/nature13695 (PMID: 25141179) applied saturation mutagenesis to find out critical mutation on BRCA1 and DBR1 protein coding sequence by HDR-based strategy using donor template library. This method based homologous recombination repair, so that the length of target region is limited. Our method employs tiling mutagenesis whose target length depends on sgRNA designed. We expand the length of target region to more than 50 kb from less than 15 kb previously reported. This is our strength compared with this report.

    The paper you raised as DOI: https://doi.org/10.1038/nbt.3450 (PMID: 25141179) applied CRISPRko tiling mutagenesis to find out critical region from 2 kb of p53 binding enhancer region by lentiviral delivery of sgRNA cassettes. Our method employs PiggyBac system that can remove the sgRNA cassettes from the chromosome with no footprint. Therefore, our method should be novel method that generates mutant library with no risk of non-targeted endogenous gene disruptions. In addition, we expand the length of target region to more than 50 kb. This is one of the advances.

    The paper you raised as DOI: https://doi.org/10.1126/science.aag2445 (PMID: 27708057) applied CRISPRi tiling mutagenesis to find out critical region from 74 kb genomic region around GATA1 and MYC by lentiviral delivery of sgRNA cassettes. Our method employs CRISPRko and PiggyBac system that can remove the sgRNA cassettes from the chromosome with no footprint. To identify combinations of critical region embedded in target regions, it would require diverse combinations of mutations or inactivation sites. To induce multiple mutations or inactivated sites, it requires multiple sgRNA cassettes integration. However, multiple integration of sgRNA cassettes have higher risk of non-targeted endogenous gene disruptions and may impair functional analysis. To eliminate the risk that integrated sgRNA cassettes disrupt non-targeted endogenous genes, it is best way to remove the sgRNA cassettes from the chromosome. Thus, to identify combinations of critical region embedded in target regions with no artifact owing to no footprint by removal of sgRNA cassettes, CRISPRko tiling mutagenesis rather than CRISPRi is better method because CRISPRi requires integrated cassettes that stably expressed sgRNA and epigenetic modifier fused to dCas9. PiggyBac system can remove the sgRNA cassettes from the chromosome with no footprint. Therefore, our method should be novel method that generates mutant library with no risk of non-targeted endogenous gene disruptions to find out combinations of critical region embedded in target regions.

    The paper you raised as DOI: https://doi.org/10.1016/j.molcel.2017.03.007 (PMID: 28416141) reported applied CRISPRi tiling mutagenesis to find out critical region from TAD scale (about 200 kb) with low magnification by lentiviral delivery of sgRNA cassettes. Our method employs PiggyBac system that can remove the sgRNA cassettes from the chromosome with no footprint. Therefore, our method should be novel method that generates mutant library with no risk of non-targeted endogenous gene disruptions.

    The paper you raised as DOI: https://doi.org/10.1038/s41588-019-0538-0 (PMID: 31784727) reported applied CRISPRi tiling mutagenesis to develop method that can find out novel regulatory element around protein coding by lentiviral delivery of sgRNA cassettes. Our method employs PiggyBac system that can remove the sgRNA cassettes from the chromosome with no footprint. Therefore, our method should be novel method that generates mutant library with no risk of non-targeted endogenous gene disruptions.

    To clarify the advantages, we will add the following sentences:

    • “To identify combinations of critical region embedded in target regions, it would require diverse combinations of mutations or inactivation sites. To induce multiple mutations or inactivated sites, it requires multiple sgRNA cassettes integration. However, multiple integration of sgRNA cassettes have higher risk of non-targeted endogenous gene disruptions and may impair functional analysis [PMID: 23435812]. To eliminate the risk that integrated sgRNA cassettes disrupt non-targeted endogenous genes, it is best way to remove the sgRNA cassettes from the chromosome. Thus, to identify combinations of critical region embedded in target regions with no artifact owing to no footprint by removal of sgRNA cassettes, CRISPRko tiling mutagenesis rather than CRISPRi is better method because CRISPRi requires integrated cassettes that stably expressed sgRNA and epigenetic modifier fused to dCas9.” in the Introduction.
    • “Here, we proposed that DNA transposon system rather than retrotransposon system is more suitable to remove sgRNA cassettes from a mutant library. Transposons are genetic elements that can relocate between genomic sites and there are two types of transposons: (1) DNA transposon is transferred by a "cut and paste" mechanism in which the transposon sequence is cut directly from the genome, and (2) retrotransposon is transferred by a "copy and paste" mechanism in which the transposon sequence is transcribed into RNA and then integrated by reverse transcribed [PMID: 21958341]. Therefore, retrotransposon is never removed from the genome. DNA transposon such as PiggyBac, Sleeping Beauty and Tol2 systems are also used as gene transfer tools in vertebrates [PMID: 26481584]. Especially, PiggyBac leaves no footprint on integrated sites after transposons relocated while other DNA transposon system leaves small insertion on integrated sites [PMID: 34064900]. In addition, excision-only-PiggyBac transposase that can remove transposons but not integrate them, is developed [PMID: 27929521]. Only PiggyBac system can remove transposons carrying sgRNA cassettes from mutant library with no footprint. Therefore, we aimed to validate PiggyBac system for CRISPRko tilling mutagenesis.” in the Introduction.
    • “CRISPRko tiling mutagenesis is conducted for less than 15 kb target genomic region so far [PMID: 26375006, 30612741], while CRISPRi tiling mutagenesis can target more than 70 kb [PMID: 27708057], it is reported that. Hence, it remains unknown unclear how length CRISPRko tiling mutagenesis could expand” in the Introduction. In addition, we are going to conduct transposon removal by exicision-only-PBase treatment with several PB mES clones, for the proof of concept that CTRL-Mutagenesis can generate mutant library with no sgRNA cassettes.

    *Reviewer #4 (Evidence, reproducibility and clarity (Required)):

    Major concerns, 1, Authors claim "to identify functionally important elements in non-coding regions in the title but there is no evidence of any functional analysis in the manuscript.*

    → Thank you very much for your suggestion, and we agree with your suggestion.

    In this paper, we just validated PiggyBac system for CRISPRko tilling mutagenesis and expanded the length of target regions.

    To change our tone that claiming usability of our method for functional analysis, we will change the following sentences:

    • Change “to identify functionally important elements in non-coding regions” to “to induce diverse combination and variety of mutations within more than 50 kb non-coding region” in the Title.
    • Add “However, not much loss-of-function screens of non-coding regulatory elements has been conducted due to ambiguous annotations compared with protein-coding genes. Tiling mutagenesis has been employed to identify critical regions embedded in non-coding regulatory elements by comparative analysis through a mutant library harbouring subtly different regions mutated within less than 15 kb region. Conventional tiling mutagenesis construct a mutant library integrated multiple sgRNA cassettes by retroviral delivery. However, multiple integration of single guide RNA (sgRNA) cassettes has higher risk of non-targeted endogenous gene disruptions and may impair functional analysis. Herein, combining tiling mutagenesis and PiggyBac transposon that can be removed with no footprint on integrated sites, we established an expanded tilling mutagenesis method named CRISPR- & Transposase-based RegionaL Mutagenesis (CTRL-Mutagenesis). We demonstrated that PiggyBac system could integrated diverse combinations and varieties of sgRNA cassettes.and then CTRL-Mutagenesis randomly induces diverse combination and variety of mutations within more than 50 kb non-coding region in murine embryonic stem cells. CTRL-Mutagenesis would apply for wider non-coding regulatory elements with no risk of non-targeted endogenous gene disruptions.” in the Abstract.
    • Delete “Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.” in the Abstract (page2, line 38-40).
    • Change “The generated random mutant mES clone library could facilitate further functional analyses of non-coding regulatory elements within the genome.” to “The generated random mutant mES clone library could develop to investigate critical regions of non-coding regulatory elements within the genome.” In the Introduction (page4, line 88-90)

    2, Genotypes of mutant library, especially Mirc56, 14,15, 16, 17 were not determined due to six tandem repeats. Thus, analysis of the relationship between genotype and biological functions is not possible. Moreover, the authors did not show any phenotypic analysis.

    → Thank you very much for your suggestion.

    The 6 tandem repeats consisted of each approximately 3.3 kb are hard to determine mutations and are uncommon.

    Therefore, we skipped genotyping *Mirc56_14, 15, 16, *and 17

    Certainly, it is drawback that we did not determine all mutations induced by CRTL-mutagenesis.

    Even so, we could determine the properties of mutant library within 37 kb genomic region from Mirc56_1 to Mirc56_13.

    Therefore, we could conclude that CTRL-mutagenesis could induce diverse combinations and variations of mutations into more than 50 kb.

    3, Multiple gRNA may cause deletion and inversion to targeted loci. With local PCR based amplification, detection of large deletion and inversion can be very difficult. I think the authors should examine and address this possibility more carefully. The definition of indel in Fig 5C should be explained in more detail.

    → Thank you very much for your comment, and we agree with your suggestion.

    We did not confirm inversion and large deletion.

    To confirm whether inversions were happened, we are going to perform PCR walking in several clones and long-read sequencing.

    4, Although the authors showed a variety of PB cassettes (Max is 17), more importantly would be to determine the actual copy number of PB cassettes. Difference between the highest and the lowest EGFP intensities in Fig 2C (Donor 300ng Effector 350ng) is approximately ~100 fold, thus ES clone bearing highest PB vector may contain ~100 copies of PB vector. PB transposon prefers insertion in active genes compared to other transposon system such as Sleeping Beauty and Tol2 transposon. (Yoshida J et al Sci Rep. 2017 Mar 2;7:43613. doi: 10.1038/srep43613.). Higher integration rates of PB vectors have a higher chance of endogenous gene disruptions and may impair functional analysis.

    → Thank you very much for your suggestion.

    We agree that cassette integration number is one important aspect of tiling mutagenesis. To determine actual copy number of PB transposon is useful information when potential user consider optimizing our method for own target region. However, to confirm whether the relationship between mutations induced and sgRNA cassettes integrated, the number of integrated cassette variety is more important because the diversity of sgRNAs variety expressed is more related to the diversity of mutations induced. Therefore, we identified the number of integrated cassette variety.

    To clarify this point, we will add we the following sentences:

    “rather than the copy number of sgRNA cassettes because the diversity of sgRNAs variety expressed is more related to the diversity of mutations induced” following to “…the number of sgRNA cassette varieties.” in the Result (page12, line 297)

    Certainly, we apologize that it is not accurate that “EGFP signal intensity correlated with the copy number of EGFP cassettes integrated into genomes[23]” in the Result (page11, line 249-250). EGFP expression levels are affected by cell cycle so that the paper reported that “Median EGFP intensities correlated with the copy number of EGFP cassettes integrated into genomes”.

    Therefore, we will delete the following sentence:

    “EGFP signal intensity correlated with the copy number of EGFP cassettes integrated into genomes[23]” in the Result (page11, line 249-250).

    To investigate how many copies our concentration of donor vector could integrate, we are going to check actual copy numbers in several clones by qPCR.

    Besides, we agree with your suggestion that “PB transposon prefers insertion in active genes compared to other transposon system such as Sleeping Beauty and Tol2 transposon. (Yoshida J et al Sci Rep. 2017 Mar 2;7:43613. doi: 10.1038/srep43613.)”

    Therefore, we will change the following sentence:

    “random TTAA sites across genomes [24]” to “random TTAA sites of transcribed region rather than intergenic region [PMID: 28252665]” in the Discussion (page14, line 357).

    However, Sleeping Beauty and Tol2 transposon remain footprint at integration sites when these transposons move [PMID: 15133768, 23143102]. Especially, SB transposon leaves canonical 5 bp insertion at integration sites so that the canonical 5bp insertion into coding sequence could disrupt the function of endogenous protein frequently. On the other hand, PB transposon remains no footprint. Therefore, excision-only-PBase can remove the PB transposon from mutant library clearly. Thus, it is no worry about that PB transposon disrupt non-targeted endogenous gene impair functional analysis if PB mutant library is treated with excision-only-PBase.

    In addition, we are going to conduct transposon removal by exicision-only-PBase treatment with several PB mES clones, for the proof of concept that CTRL-Mutagenesis can generate mutant library with no sgRNA cassettes.

    5, Most non-coding regions are located at autosomes. Genotyping would be very difficult or even impossible by the current PCR based strategy.

    → Thank you very much for your comment, and we agree with your suggestion.

    This is one of our issues.

    We expect that CTRL-Mutagenesis could be valid on other biallelic locus.

    Therefore, we raised predicted issue such as complex genotyping and proposed one solution.

    When we target other biallelic locus, we must determine whether the combination of mutations induced are cis- or trans-mutations. Haplotype phasing, combined long-read sequencing with SNP markers within ROI on maternal/paternal chromosome, assembles each allele via SNP markers on each read [PMID: 35710642]. Therefore, combining CTRL-Mutagenesis on heterozygotic alleles of cells derived from such as human or murine hybrid with haplotype phasing might simplify genotyping.

    We will add the following sentences:

    “In this study, CTRL-Mutagenesis was validated by genotyping on mono allele in male mES cells to avoid investigating whether the combination of mutations induced are cis- or trans-mutations. All genotypes on Mirc56 should be hemizygous and these mutations induced might be cis-mutations so that we determined the genotypes by amplifying approximately 200 bp around the target sites. However, we did not confirm large mutations such as deletion of the genomic region between target sites and inversion. Long-read sequencing might capture their large mutations. Besides, we also expect that CTRL-Mutagenesis could be valid for ROI on biallelic autosome and X chromosome in female. Therefore, it is required to determine whether the combination of mutations induced are cis- or trans-mutation. Haplotype phasing, combined long-read sequencing with SNP markers within ROI on maternal/paternal chromosome, assembles each allele via SNP markers on each read [PMID: 35710642]. Therefore, combining CTRL-Mutagenesis on heterozygotic alleles of cells derived from such as human or murine hybrid with haplotype phasing might simplify genotyping.” in the Discussion.

    Moreover, genome-wide NGS and nanopore Cas9-treated sequencing (nCATs) could also help us to read the mutations without PCR-amplification. However, both methods can obtain reads of target regions with low frequency. Therefore, it is difficult to perform multiplex samples for mutant library.

    *6, Fig 4C, large amounts of Cas9 independent EGFP positive cells suggest the current system is not efficient. *

    → Thank you very much for your comment.

    We cannot agree with your indication.

    In fact, by the cutoff set in Cas9-untreated cells, the EGxxFP system successfully selected at least 76 mutant clones (87.4%) harboring mutations within Mirc56_1 to Mirc56_13. Moreover, we could seed 180 single-cells for single cloning by FACS once.

    To enhance this point, we added the following sentences:

    “Moreover, at least 76 out of 87 PB mES clones have mutations within all analysed Mirc56_Xs (Figure 5C). Therefore, the EGxxFP system could selected ROI mutant mES clones efficiently.” following to “…depended on integrated sgRNA cassettes.” in the Discussion (page13, line 355)

    *Reviewer #4 (Significance (Required)):

    The authors claim "Functional analysis" in the manuscript title but there is no evidence of functional analysis in the manuscript.*

    → Thank you very much for your suggestion, and we agree with your suggestion.

    In this paper, we just validated PiggyBac system for CRISPRko tilling mutagenesis and expanded the length of target regions.

    To change our tone that claiming usability of our method for functional analysis, we will change the following sentences:

    • Change “to identify functionally important elements in non-coding regions” to “to induce diverse combination and variety of mutations within more than 50 kb non-coding region” in the Title.
    • Add “However, not much loss-of-function screens of non-coding regulatory elements has been conducted due to ambiguous annotations compared with protein-coding genes. Tiling mutagenesis has been employed to identify critical regions embedded in non-coding regulatory elements by comparative analysis through a mutant library harbouring subtly different regions mutated within less than 15 kb region. Conventional tiling mutagenesis construct a mutant library integrated multiple sgRNA cassettes by retroviral delivery. However, multiple integration of single guide RNA (sgRNA) cassettes has higher risk of non-targeted endogenous gene disruptions and may impair functional analysis. Herein, combining tiling mutagenesis and PiggyBac transposon that can be removed with no footprint on integrated sites, we established an expanded tilling mutagenesis method named CRISPR- & Transposase-based RegionaL Mutagenesis (CTRL-Mutagenesis). We demonstrated that PiggyBac system could integrated diverse combinations and varieties of sgRNA cassettes.and then CTRL-Mutagenesis randomly induces diverse combination and variety of mutations within more than 50 kb non-coding region in murine embryonic stem cells. CTRL-Mutagenesis would apply for wider non-coding regulatory elements with no risk of non-targeted endogenous gene disruptions.” in the Abstract.
    • Delete “Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.” in the Abstract (page2, line 38-40).
    • Change “The generated random mutant mES clone library could facilitate further functional analyses of non-coding regulatory elements within the genome.” to “The generated random mutant mES clone library could develop to investigate critical regions of non-coding regulatory elements within the genome.” In the Introduction (page4, line 88-90).
  2. Review Commons

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    Referee #4

    Evidence, reproducibility and clarity

    Morimoto et al. presented a regional random mutagenesis study using multiple sgRNAs in PiggyBac transposon vectors to analyze the relationship between genotype and biological functions. For proof of principle, authors chose the X-linked miRNA cluster Mirc56 and made its mutant library.

    Major concerns

    1. Authors claim "to identify functionally important elements in non-coding regions in the title but there is no evidence of any functional analysis in the manuscript.
    2. Genotypes of mutant library, especially Mirc56, 14,15, 16, 17 were not determined due to six tandem repeats. Thus, analysis of the relationship between genotype and biological functions is not possible. Moreover, the authors did not show any phenotypic analysis.
    3. Multiple gRNA may cause deletion and inversion to targeted loci. With local PCR based amplification, detection of large deletion and inversion can be very difficult. I think the authors should examine and address this possibility more carefully. The definition of indel in Fig 5C should be explained in more detail.
    4. Although the authors showed a variety of PB cassettes (Max is 17), more importantly would be to determine the actual copy number of PB cassettes. Difference between the highest and the lowest EGFP intensities in Fig 2C (Donor 300ng Effector 350ng) is approximately ~100 fold, thus ES clone bearing highest PB vector may contain ~100 copies of PB vector. PB transposon prefers insertion in active genes compared to other transposon system such as Sleeping Beauty and Tol2 transposon. (Yoshida J et al Sci Rep. 2017 Mar 2;7:43613. doi: 10.1038/srep43613.). Higher integration rates of PB vectors have a higher chance of endogenous gene disruptions and may impair functional analysis.
    5. Most non-coding regions are located at autosomes. Genotyping would be very difficult or even impossible by the current PCR based strategy.
    6. Fig 4C, large amounts of Cas9 independent EGFP positive cells suggest the current system is not efficient.

    Significance

    The authors claim "Functional analysis" in the manuscript title but there is no evidence of functional analysis in the manuscript.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The study proposes a method utilizing EGxxFP, piggybac, and CRISPR-Cas9 systems to generate a random mutation library in non-coding genomic regions, such as cis-element regions or microRNA clusters. As a proof-of-concept of this method, a random mutation library of Mirc56 microRNA cluster was generated. The manuscript highlights the creation of regional mutations using mES cells.

    Major comments:

    Authors claim that "CTRL-mutagenesis randomly induces diverse mutations only within the targeted regions in murine embryonic stem (mES) cells.", however, the outcome of mutations is not entirely random since most of the mutations are regional deletions. For example, despite the random distribution of gRNAs per cell, the inner regions like Mirc56_5 or Mirc56_8 are mutated with >80% efficiency. Also, although the authors discuss that the lower mutation frequency observed for Mirc56_2 and 4 may be due to a technical error, confirming this by repeating the experiment would be important to prove the usability of this method. Additionally, the experiments were performed on the haploid X chromosome of a male cell line. It is questionable whether this method can be generalized to other regions located in the other chromosomes. Clarifying These points would be essential especially because the focus of this manuscript is to describe the efficiency of this novel methodology.

    The limitations of the methods seem not to be fully described in the manuscript and must be clarified. Compared to the previous studies (see "significance" section for details), this method is inferior in that (1) it is time-consuming because it requires clonal expansion of single cells and (2) it has low throughput because it requires genome sequencing due to the occurrence of deletions. These points should be described for the potential users of this methodology. For example, it may be useful to detail the time consumption in each experimental step in Fig. 4A.

    Minor comments:

    Data and methods are well-presented for reproducibility. The EGFP-positive ratio may be added to Fig. 4C for clarity.

    Enhance referencing accuracy, rectify DOI format in ref 21, and ensure consistency in citation formatting, e.g., ref 32.

    It seems that the experimental condition (e.g. The amount of vectors used for transfection) should be re-considered every time the researcher wants to set up an experiment changing target genomic regions, cell types etc. If so, this also should be described in the text for potential users of this method.

    Referees cross-commenting

    Major concerns raised by the reviewers, including the non-random nature of mutations, challenges in library resolution, and unclear advantages over existing methodologies, all seem to be reasonable. These concerns should be addressed before publication, especially because this is a methodology paper that reports the usability of this novel methodology.

    Significance

    The method combines CRISPR screens with EGxxFP and PiggyBac systems for complex cell library generation, albeit with limitations in throughput and time consumption.

    There were various methods described in the late 2010's which aimed to screen for the functional non-coding regions using approaches such as KO-based, HDR-based, and epigenetic silencing using dCas9 (for example, PMID: 25141179, 26751173, 27708057, 28416141, 31784727). The authors should summarize what would be the strength of their method compared to these previously described methodologies. The strength of this methodology seems to be moderate complexity and cost-effectiveness compared to these previous techniques. It may be difficult for this methodology to become a state-of-the-art method to evaluate cis-element combinations, but it can be beneficial to researchers wanting to set up a low-cost system that can produce moderately complex cell libraries.

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    Referee #2

    Evidence, reproducibility and clarity

    In this paper, Morimoto et al. present CTRL-Mutagenesis, a method for region-specific random mutagenesis, aiming to identify functionally important elements in non-coding regions of the genome. While the idea to integrate sgRNA in the genome by the PiggyBack system is interesting there are several major concerns that limit the novelty and impact of this study as outlined below.

    Major concerns:

    1. Concern about the Novelty of Functional Analysis Platforms: The authors claim that there are no established platforms for the study of cis-elements or microRNA clusters. This assertion seems inaccurate, as previous studies have utilized Cas9 tiling screens to investigate cis-regulatory elements (CREs) and large-scale screens to probe microRNA functions, as exemplified by the works of Canver et al. in Nature 2015, Gasperini et al. in Cell 2019, and others.
    2. Advantages of PiggyBack System Over Lentiviral Integration: The paper does not clearly articulate the advantages of their proposed PiggyBack-based system for sgRNA integration over traditional lentiviral integration. Both methods facilitate the random integration of multiple gRNAs, but the paper lacks a comparative analysis or justification for choosing the PiggyBack system.
    3. Lack of Comparative Analysis with Alternative Methods: The authors did not provide a comparison of CTRL-Mutagenesis with other existing screening methods. Such a comparison is crucial for understanding the effectiveness and efficiency of the new method in relation to established techniques.
    4. Limitations in Library Resolution: The paper acknowledges the limited resolution of their proposed library. The authors might have explored the use of base editors for enhanced resolution in such screens, as base editing could potentially offer more precise and controlled mutagenesis as briefly mentioned in the discussion.
    5. Absence of Functional Data Post-Mutagenesis: A significant limitation of the study is the absence of functional data following the creation of cells with different mutations. While the authors speculate about using differentiation systems or organoids for practical applications, they do not provide empirical data to demonstrate the utility of the CTRL-Mutagenesis approach. This lack of functional validation raises questions about the practical applicability of the method.

    Significance

    In summary, while the idea to integrate sgRNA in the genome by the PiggyBack system is interesting the claim of novelty is questionable due to existing methods in the field. The advantages of their system over existing technologies are not clearly articulated, and a lack of comparative analysis with other methods leaves the efficiency of CTRL-Mutagenesis uncertain. Moreover, the limited resolution of their library and the absence of functional data post-mutagenesis are significant drawbacks that need to be addressed in future research to ascertain the method's practical utility.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In "Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions", Morimoto et al. detail a method for generating clonal cell lines containing mutations tiled across a region of interest. Briefly, they create a pool of sgRNAs tiling a region of interest, then utilize the PiggyBac transposase to insert random sgRNAs from this pool into cellular DNA. Cas9 cleavage directed by cellular sgRNAs creates small indels and large deletions in cells that can be grown out into clonal cell lines. They apply their method to study the Mirc56 microRNA cluster to generate 87 clones, each clone being edited by an average of 4.7/17 sgRNAs. The authors suggest that these cell lines could be used to elucidate the functional importance of non-coding regions.

    Major comments:

    1. Mirc56_2 and 4 showed lower integration rates, and the authors suggest that this could be due to sgRNA pool imbalance. The authors should validate this by performing sequencing of the input sgRNA and cassettes.
    2. Clonal analysis in Figure 5c is unclear
      • a. Figure 5c indicates that all changes were homozygous (e.g. both alleles were deleted). Was this the case in all clones? Or were some mutations heterozygous?
      • b. Many clones in Figure 5c show that the entire region was deleted (all black dots). Could this be due to some experimental error or misinterpretation of the sequencing data, or could it be validated using some orthogonal method? This is especially surprising for clones in which the final guide (Mirc56_13) was not detected yet the final site (Mirc56_13) was reported as "Regional deletion".
    3. Next-generation targeted sequencing of clones should be made publicly accessible.
    4. OPTIONAL - Cassette integration number is understudied. One important aspect of tiling mutagenesis is the control over how many guides are present in each cell. The authors report an average of 4.7 cassettes/cell. This could be modulated by the amount of donor vector added, and indeed the authors performed titration experiments, but only with a fluorescent reporter readout. It would be very useful to know how the concentration of donor vector corresponds to the number of cassettes/cell - perhaps genotyping of clones from one or two additional experiments would be sufficient.

    Minor comments:

    1. The background fails to acknowledge the work of CRISPR-Cas tiling screens (e.g. https://doi-org/10.1038/nbt.3450) or CRISPR-Cas in creating mutagenesis in cell lines (e.g. https://doi-org/10.1007/978-1-0716-0247-8_29)
    2. Figure 1 left 'ROI random mutant PB mES cell' should be horizontally aligned so Mir_1, Mir_2 and MirX align with the upper figure.
    3. It is interesting and unexpected that some guides never induce indels, even in the absence of a regional deletion (e.g. Mirc56_3, Mirc56_7). Why might this be? Was there perhaps an error in the assignment of these guides to these cells?
    4. Regarding Mirc56_2 and 4 integration, on line 34 the authors suggest that "We suspect this was caused by a technical error, such as an unequal amount of sgRNA donor vector or the sequence in sgRNA cassettes affecting integration efficiency or cell growth." sgRNA library imbalance would be a technical error, but integration affecting cell growth is not a technical error. This sentence should be reworded.
    5. Line 540 "ration" is the incorrect word - perhaps "ratio"?
    6. Plot 5b should be shown as a histogram rather than a swarm plot to show how many clones were in each category.

    Referees cross-commenting

    All reviewers note previous functional non-coding screens and the lack of justification of the author's PiggyBac system. The authors should consider strengthening the justification and adding comparisons to existing methods if future revisions are considered.

    Significance

    General assessment: The authors are successful in creating clonal cell lines bearing a variety of mutations. Unfortunately, the cell lines also have transposase-mediated insertion events of the sgRNA cassettes at unknown positions in the genome, which will hamper the interpretability of any experiment using these cell lines. The authors fail to justify the use of the transposase and integration of the sgRNA, especially compared to lentiviral transfection or RNPs which would produce edits at the region of interest. Alternately, integrated sgRNA cassettes could have been excised with Flp recombinase as in https://doi-org/10.1007/978-1-0716-0247-8_29. Additionally, the genotyping analysis is unclear, and seems to indicate that each clone bears homozygous mutations, with several clones showing deletions of the entire region.

    Advance: The authors are motivated to create clones using tiling mutagenesis. Tiling mutagenesis has already been performed without transposases (e.g. https://doi-org/10.1038/nbt.3450, https://doi.org/10.1371/journal.pone.0170445, https://doi.org/10.1038/s41467-019-12489-8) in the context of a screen, and clones have already been created using CRISPR/Cas9 mutagenesis so the advance presented in this manuscript over previous published work is unclear.

    Audience: The manuscript is written for the basic research audience, and the method could be applied to the study of regions of interest in many diseases. However, the unexcised use of transposases make the method less desirable than other methods.

    I am a computational biologist with experience in CRISPR tiling screens and CRISPR amplicon sequencing analysis.

  6. Version published to 10.1101/2023.09.12.557308v2 on bioRxiv
  7. Version published to 10.1101/2023.09.12.557308v1 on bioRxiv