ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes
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Abstract
Bacterial artificial chromosome (BAC)–based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. Although homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here, we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call exogenous/synthetic intronization (ESI) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.
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Reply to the reviewers
We thank the reviewers for their comments and outline below how we plan to address them.
*------------------------------------------------------------------------------ ** Reviewer #1 (Evidence, reproducibility and clarity (Required)):
**Summary:**
Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). The authors here describe a method to modify bacterial artificial chromosomes (BAC) harbouring gene loci from eukaryotes. When wanting to modify a BAC an antibiotic selection cassette is often included alongside the desired mutation/modification to increase the number of successful …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
We thank the reviewers for their comments and outline below how we plan to address them.
*------------------------------------------------------------------------------ ** Reviewer #1 (Evidence, reproducibility and clarity (Required)):
**Summary:**
Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). The authors here describe a method to modify bacterial artificial chromosomes (BAC) harbouring gene loci from eukaryotes. When wanting to modify a BAC an antibiotic selection cassette is often included alongside the desired mutation/modification to increase the number of successful recombinants in E.coli. Traditionally, this is removed in a second recombination process to leave only the desired modification. The novelty in the procedure described herein is to add a synthetic intron consensus sequence around the selection cassette, which eliminates the need for the subsequent removal of the antibiotic cassette from the BAC before transfection into mammalian cells, saving time and resources. The technique is clever in its simplicity and appears to function for a number of gene loci. The authors validated the correct functioning of the modified BACs for a number of genes using three main assays - transcript level, protein level and localisation.
**Major comments:**
*Are the key conclusions convincing?*
The conclusion that the method described generates functional modified BACs is valid.
*Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?*
While the method is successfully employed in this study, its efficiency is not quantified in relation to the state-of-the-art as described in the introduction. One assumes it would be more efficient, but this has not been tested empirically in the paper. Does the inclusion of the synthetic intron sequence have an effect on the efficiency of modifying BACs compared to a more typical two-step positive/negative antibiotic selection cassette? *
This is a good point that we did not directly address. In general, the efficiency is similar to that of integrating any cassette with selectable marker, as has been published (Poser et al 2008), and therefore also higher than the two-step counterselection method, which requires such a cassette integration in the first step alone. We will include new data specifically addressing the efficiency of our new method (see specifics below)
The functionality of this approach rests entirely on the ability of the target cell to correctly splice out the synthetic intron. The authors are aware of this potential problem as highlighted in the lines below, but do not make efforts to explicitly test splicing. On lines 224-225, the authors state "We cannot exclude that a small portion of synthetic introns within individual cells are misspliced". On lines 230-231 it is stated that "mis-spliced mRNAs are probably minimal and degraded by nonsense-mediated decay". On lines 215-217, the authors describe an "investigation of transgenic lines at the single-cell level" that suggests "the synthetic intron is correctly spliced out in all the cells of the population". How do the authors reach this conclusion? U2OS and HeLa cells are considered very "robust" and may not show detectable consequences when stressed with an increased level of nonsense-mediated decay. Further, many genes maintain a high level of expression that buffers them against small changes in transcription/splicing. The synthetic intron might have a bigger impact on more tightly regulated genes, so assessing the splicing rate would be essential if the authors wish to advocate their technique as generally applicable. *
We will assay for splicing efficiency as outlined below.
The ability of the synthetic intron to be removed from final transcripts depends on functioning splicing machinery. The authors might emphasise this issue, as spliceosome mutations are important fields of study and might not be compatible with this method. *
We can add this in the text
The authors used un-directed integration of each BAC under study. Therefore, it is hard to assess what effect the synthetic intron has, as the authors only ever assess the downstream levels of the correctly spliced, translated and localised protein. The authors themselves state that this can lead to clonal variations in expression of up to 2-fold and on line 250 that this variation "could compensate for synthetic intron effects", but make no effort to test this. Again, lines 267-268 highlight the potential dangers of potential effects of the synthetic introns, but do not test these.
*Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.*
If not already performed, a large number of bacterial colonies should be screened for the correct modification and frequency of correct ones reported. This frequency - reported for at least three different modifications - would estimate what sort of efficiency this method provides. The modified region of each BAC should be sequenced and the results reported. The rate of exactly modified clones is important, in case of spontaneous or low fidelity integration of the antibiotic cassette. The percentage of transcripts that have the synthetic intron correctly spliced out should be measured for some of the BAC constructs used in the study. A direct head-to-head comparison of this newer method compared to other techniques, or even the authors' own previous two-step approach is necessary to assess the benefits of this method. Preferably, the experiment would be run in parallel with and without antibiotic selection applied, to show that it drastically improves chances of finding a correct clone.
We will generate 3 new mutations in BACs and analyze both the efficiency of integration by PCR and accuracy via sequencing. In practice, we have observed that the efficiency is similar to any other cassette integration, such as a GFP tag (Poser et al Nature Methods 2008) or a counterselection cassette (Bird et al Nature Methods 2012) (80-90%). Integrating a mutation via the second step of the counterselection method introduces a further 20% decrease in efficiencies on average.
*Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.*
Repeating the transformation of the BAC and targeting cassette and assessing the recombination efficiency and sequencing should only require existing reagents and take less than a week or two to complete. Quantitative RT-PCR to assess the percentage of transcripts that have the synthetic intron spliced out would take a little more work. However, this should not be a considerable investment in time or resources for a standard microbiology laboratory and could be completed within a few weeks using modern techniques, such as that described in Londoño et al. 2016. Repeating all the experiments in parallel would be considerable work and would only be strictly necessary if the authors wish to emphasise the benefits of their method over the many others already in wide use. *
We will use quantitative PCR to estimate the fraction of transcripts that correctly splice out the artificial intron for two clonal cell lines characterized in the study: RNAi-resistant AurA-GFP (Fig 4), and GTSE1-14A (newly introduced; see below). While the exact method described in Londoño et al 2016 will not be applicable due to the larger size of the artificial intron, we believe we can adapt it to detect different splicing events. *
*Are the data and the methods presented in such a way that they can be reproduced?*
Barring the omission of Table S1, which presumably includes exact information on the BACs modified and sequences used etc., there is sufficient other data and methods to allow the experiments to be repeated. Targeting the ESI procedure to the middle of exons is likely to have a bigger impact for smaller exons as the authors mention on lines 99-100. Making it clear which exon sizes for each gene were successfully targeted in this study would help give some idea of how significant a problem this might be. Perhaps Table S1 contains this information, but it was not provided. It would also help reviewers check the design strategies.
We apologize for inadvertently failing to upload Table S1 on bioRxiv. It has been uploaded now as part of this submission process. This table indeed contains BAC and target sequence information, including the size of the targeted exon (and the 2 “new” resulting exons). Targeted exons range in size from 138bp to 1537bp, and “new” exons are as small as 48bp.
*Are the experiments adequately replicated and statistical analysis adequate?*
The replication and statistically analysis of the data as presented appear adequate. Figure Legends should state the statistic used to generate error bars.
This will be updated
**Minor comments:**
Specific experimental issues that are easily addressable. Are the promoters used in the vectors described universally functional? For example, is the PGK promoter functional in yeast? *
The PGK promoter contained in the cassettes is a mammalian promoter, which has also been reported to work in flies.
*Are prior studies referenced appropriately?*
The manuscript may benefit from the referencing of BAC modification techniques from a wider variety of groups, such as those using CRISPR-guided recombineering (Pyne et al. 2015). *
*We will add citations of more techniques
*Are the text and figures clear and accurate?*
The body text is very clear save minor typographical or grammatical errors. Regarding figures, some of the coloured text in Figure 1 is somewhat illegible when printed in grayscale.
Line 278 - The acronyms LAP and NLAP are not defined/explained.
Antibody section starting Line 282 may fit better next to Western Blot section.
Figure 2C - The blot images would benefit from arrows to indicate expected sizes of proteins.
Figure 3A - the graph may benefit from a dashed line at 100% to highlight that values are normalised to controls.
Figure 4 - The differences between panels B & C are unclear.
Figure 4E - The legend could provide a little more detail on cell cycle stage/status of the captured cells.
All of the above will be addressed accordingly
*Do you have suggestions that would help the authors improve the presentation of their data and conclusions?*
Lines 23-27 are somewhat unclear and feel out of context. Perhaps the authors could clarify this as a further advantage of using BACs instead of endogenous gene modifications. *
*Thanks for the input, we will clarify this.
While not affecting the factual content of the paper, I would advocate that the authors format the method described in Figure S3 into a more detailed text based layout similar to that seen in a typical Nature Methods article. However, this may depend on the format required by any eventual publishing journal. *
We prefer the graphical protocol, but will discuss whether to add a text protocol with the journal editor.
That all of the work the paper was carried out in human cell lines and using human genes is a further caveat, but the authors admit this in the discussion and one would assume that most mammalian cells would respond similarly in their ability to splice out the synthetic intron.
Reviewer #1 (Significance (Required)):
*Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.*
This work is a formal description of a newer method that could be useful for many of those employing bacterial artificial chromosomes in numerous studies, such as gene regulation.
*Place the work in the context of the existing literature (provide references, where appropriate).*
This work builds on methodology previously published by the authors - a counter-selection two-step procedure (Bird et al. 2011). It sets out to formally describe a method merely mentioned as "BAC intronization" in a later paper by some of the authors (Zheng et al. 2014). Other alternative one-step procedures are also available, but present a different set of challenges (Lyozin et al. 2014). Some newer approaches, such as those using CRISPR-guided recombineering (Pyne et al. 2015) or systems that combine CRISPR and positive/negative selection cassettes (Wang et al. 2016) may be slightly more efficient, but are also more complex in their design.
Bird et al. 2011 DOI: 10/dv776q
Pyne et al. 2015 DOI: 10/f7jx92
Wang et al. 2016 DOI: 10/f89db5
Zheng et al. 2014 DOI: 10/f5pkr6
*State what audience might be interested in and influenced by the reported findings.*
As a technology paper this work should have interest from a broad field of research. While the use of BACs could sometimes be considered more traditional in light of the explosion in CRISPR-based genome editing capabilities, it is definitely seeing a resurgence as the limitations of CRISPR in modifying large regions of genome become more apparent. Therefore, technologies that accelerate the modification of BACs could prove increasingly useful. As category of audience, all those involved in significant recombineering or gene/genome engineering would potentially benefit.
*Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.*
Synthetic genomics, synthetic biology, cancer cell biology, gene and genome engineering
REFEREES CROSS COMMENTING
I would agree with reviewer two's assessment that we both view the paper in a similar light.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
This is a methods-focused paper that presents a strategy to efficiently introduce mutations into a bacterial artificial transgene using synthetic introns. BAC-based methods have been an effective strategy for introducing trans genes into human cells to achieve near-endogenous expression, including extensive work from these authors. However, generating mutations and changes within the internal coding sequence presents some challenges for how to target these mutations and select for the mutated form. Here, the authors describe a way to overcome this by introducing synthetic introns into an adjacent sequence. This allows them to introduce a selectable marker and conduct the molecular biology without creating complications downstream for the functionality of the protein.
This method is carefully described and presented. The authors also provide clear validation by using this to create RNAi-resistant versions of multiple different mitotic factors as well as creating targeted mutants that alter the functional properties of a protein. This work clearly takes advantage of other ongoing studies from these labs (including mutants and cell lines that appear to also have been described elsewhere), but the ability to combine these in a single paper and clearly describe the method provides a helpful advance and validation.
Based on the description and data presented, I think that things are clear and carefully validated. As such, I do not have technical comments or concerns and I would be comfortable with this paper appearing in an appropriate journal in its present form.
Reviewer #2 (Significance (Required)):
This is a solid methods paper, but for considering the nature of the impact and significance of this paper, there are several things to note:
1.The BAC-based method does appear to be a powerful and effective strategy. However, beyond the work of Mitocheck and the authors that are part of this paper, this has not seen widespread adoption. It is possible that this current method may increase its usage due to the value of the targeted mutations within the coding sequence, but at present it is not a broadly used strategy.
We agree that using BACs as transgenes has not seen widespread adoption as a tool on the broader cell biology community (although certainly beyond members of the Mitocheck consortium). This is likely because many erroneously think that it is a technique for specialist laboratories. We are trying to change this! For reasons outlined below, there is still an increasing desire for conditional analysis of mutated genes under physiological expression/regulation frequently not attainable via directed Cas9-based mutation. A major aim of this paper is thus to further simplify the methods for generating modified BAC transgenes.
2.This BAC-based approach (and also RNAi) are becoming increasingly replaced by the use of CRISPR/Cas9 genome editing. The absence of Cas9-based strategies in this paper limits the potential impact and reach of this paper. The authors do mention the possibility of using a similar synthetic intron strategy for use with Cas9 in the Discussion, and appear to have conducted some experiments. If possible, it would substantially increase the value of this paper if this data and strategy were also included in the Results section (acknowledging that this may still be a work in progress). *
While some uses of BAC transgenes are in some cases better replaced by CRISPR/Cas9 techniques (i.e. GFP tagging), there are several occasions where using BACs are preferable: As stated in the text, RNAi-resistant BACs allow for conditional analysis of recessive mutations. Mutations in essential genes that are lethal will prevent growth and recovery of viable cells if integrated into the genome via Cas9. Additionally, deleterious mutations are prone to accumulate suppressive changes in chromosome integrity or gene expression during the procedure of selecting and expanding Cas9-modified cells for analysis, particularly in the genomically instable cancer cell lines frequently employed.
We use both BACs and CRISPR/Cas9 in our lab according to our needs.
We do have an ongoing project to apply this intronization technique to enable more efficient selection of CRISPR/Cas9 integrations. Preliminary results suggest that it works to allow selection of point mutations, but it is still being optimized, including a redesign of the cassette, and is not ready for publication.
3.The method is solid and well-validated, but there are no new results or insights presented in this paper from the work that is described (this is fine, just commenting for considering the right journal fit).
As “biological insights” gained as a result of this technique we had cited a couple studies that made use of the technique already (to functionally analyze a microcephaly-associated mutation in the centriolar protein CPAP at the single cell level in HeLa cells and neural progenitor cells (Zheng et al 2014, Gabirel et al 2016)). As a response to this critique to include “new biology” in this paper, we will add new unpublished data investigating a specific question: Is the cell-cycle-regulated disruption of the EB1-GTSE1 (microtubule plus-end tracking proteins) interaction in mitosis required for chromosome segregation fidelity? We have generated a GTSE1 mutant with 14 phosphosites mutated to alanine using this technique. We will present the effect on chromosome segregation.
REFEREES CROSS COMMENTING
It appears that both reviewers are largely on the same page regarding this paper. *
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Referee #2
Evidence, reproducibility and clarity
This is a methods-focused paper that presents a strategy to efficiently introduce mutations into a bacterial artificial transgene using synthetic introns. BAC-based methods have been an effective strategy for introducing trans genes into human cells to achieve near-endogenous expression, including extensive work from these authors. However, generating mutations and changes within the internal coding sequence presents some challenges for how to target these mutations and select for the mutated form. Here, the authors describe a way to overcome this by introducing synthetic introns into an adjacent sequence. This allows them to …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This is a methods-focused paper that presents a strategy to efficiently introduce mutations into a bacterial artificial transgene using synthetic introns. BAC-based methods have been an effective strategy for introducing trans genes into human cells to achieve near-endogenous expression, including extensive work from these authors. However, generating mutations and changes within the internal coding sequence presents some challenges for how to target these mutations and select for the mutated form. Here, the authors describe a way to overcome this by introducing synthetic introns into an adjacent sequence. This allows them to introduce a selectable marker and conduct the molecular biology without creating complications downstream for the functionality of the protein.
This method is carefully described and presented. The authors also provide clear validation by using this to create RNAi-resistant versions of multiple different mitotic factors as well as creating targeted mutants that alter the functional properties of a protein. This work clearly takes advantage of other ongoing studies from these labs (including mutants and cell lines that appear to also have been described elsewhere), but the ability to combine these in a single paper and clearly describe the method provides a helpful advance and validation.
Based on the description and data presented, I think that things are clear and carefully validated. As such, I do not have technical comments or concerns and I would be comfortable with this paper appearing in an appropriate journal in its present form.
Significance
This is a solid methods paper, but for considering the nature of the impact and significance of this paper, there are several things to note:
1.The BAC-based method does appear to be a powerful and effective strategy. However, beyond the work of Mitocheck and the authors that are part of this paper, this has not seen widespread adoption. It is possible that this current method may increase its usage due to the value of the targeted mutations within the coding sequence, but at present it is not a broadly used strategy.
2.This BAC-based approach (and also RNAi) are becoming increasingly replaced by the use of CRISPR/Cas9 genome editing. The absence of Cas9-based strategies in this paper limits the potential impact and reach of this paper. The authors do mention the possibility of using a similar synthetic intron strategy for use with Cas9 in the Discussion, and appear to have conducted some experiments. If possible, it would substantially increase the value of this paper if this data and strategy were also included in the Results section (acknowledging that this may still be a work in progress).
3.The method is solid and well-validated, but there are no new results or insights presented in this paper from the work that is described (this is fine, just commenting for considering the right journal fit).
REFEREES CROSS COMMENTING
It appears that both reviewers are largely on the same page regarding this paper.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary:
Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). The authors here describe a method to modify bacterial artificial chromosomes (BAC) harbouring gene loci from eukaryotes. When wanting to modify a BAC an antibiotic selection cassette is often included alongside the desired mutation/modification to increase the number of successful recombinants in E.coli. Traditionally, this is removed in a second recombination process to leave only the desired modification. The novelty in the procedure described herein is to add a synthetic intron consensus sequence …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary:
Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). The authors here describe a method to modify bacterial artificial chromosomes (BAC) harbouring gene loci from eukaryotes. When wanting to modify a BAC an antibiotic selection cassette is often included alongside the desired mutation/modification to increase the number of successful recombinants in E.coli. Traditionally, this is removed in a second recombination process to leave only the desired modification. The novelty in the procedure described herein is to add a synthetic intron consensus sequence around the selection cassette, which eliminates the need for the subsequent removal of the antibiotic cassette from the BAC before transfection into mammalian cells, saving time and resources. The technique is clever in its simplicity and appears to function for a number of gene loci. The authors validated the correct functioning of the modified BACs for a number of genes using three main assays - transcript level, protein level and localisation.
Major comments:
Are the key conclusions convincing?
The conclusion that the method described generates functional modified BACs is valid.
Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
While the method is successfully employed in this study, its efficiency is not quantified in relation to the state-of-the-art as described in the introduction. One assumes it would be more efficient, but this has not been tested empirically in the paper. Does the inclusion of the synthetic intron sequence have an effect on the efficiency of modifying BACs compared to a more typical two-step positive/negative antibiotic selection cassette? The functionality of this approach rests entirely on the ability of the target cell to correctly splice out the synthetic intron. The authors are aware of this potential problem as highlighted in the lines below, but do not make efforts to explicitly test splicing. On lines 224-225, the authors state "We cannot exclude that a small portion of synthetic introns within individual cells are misspliced". On lines 230-231 it is stated that "mis-spliced mRNAs are probably minimal and degraded by nonsense-mediated decay". On lines 215-217, the authors describe an "investigation of transgenic lines at the single-cell level" that suggests "the synthetic intron is correctly spliced out in all the cells of the population". How do the authors reach this conclusion? U2OS and HeLa cells are considered very "robust" and may not show detectable consequences when stressed with an increased level of nonsense-mediated decay. Further, many genes maintain a high level of expression that buffers them against small changes in transcription/splicing. The synthetic intron might have a bigger impact on more tightly regulated genes, so assessing the splicing rate would be essential if the authors wish to advocate their technique as generally applicable. The ability of the synthetic intron to be removed from final transcripts depends on functioning splicing machinery. The authors might emphasise this issue, as spliceosome mutations are important fields of study and might not be compatible with this method. The authors used un-directed integration of each BAC under study. Therefore, it is hard to assess what effect the synthetic intron has, as the authors only ever assess the downstream levels of the correctly spliced, translated and localised protein. The authors themselves state that this can lead to clonal variations in expression of up to 2-fold and on line 250 that this variation "could compensate for synthetic intron effects", but make no effort to test this. Again, lines 267-268 highlight the potential dangers of potential effects of the synthetic introns, but do not test these.
Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.
If not already performed, a large number of bacterial colonies should be screened for the correct modification and frequency of correct ones reported. This frequency - reported for at least three different modifications - would estimate what sort of efficiency this method provides. The modified region of each BAC should be sequenced and the results reported. The rate of exactly modified clones is important, in case of spontaneous or low fidelity integration of the antibiotic cassette. The percentage of transcripts that have the synthetic intron correctly spliced out should be measured for some of the BAC constructs used in the study. A direct head-to-head comparison of this newer method compared to other techniques, or even the authors' own previous two-step approach is necessary to assess the benefits of this method. Preferably, the experiment would be run in parallel with and without antibiotic selection applied, to show that it drastically improves chances of finding a correct clone.
Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
Repeating the transformation of the BAC and targeting cassette and assessing the recombination efficiency and sequencing should only require existing reagents and take less than a week or two to complete. Quantitative RT-PCR to assess the percentage of transcripts that have the synthetic intron spliced out would take a little more work. However, this should not be a considerable investment in time or resources for a standard microbiology laboratory and could be completed within a few weeks using modern techniques, such as that described in Londoño et al. 2016. Repeating all the experiments in parallel would be considerable work and would only be strictly necessary if the authors wish to emphasise the benefits of their method over the many others already in wide use.
Are the data and the methods presented in such a way that they can be reproduced?
Barring the omission of Table S1, which presumably includes exact information on the BACs modified and sequences used etc., there is sufficient other data and methods to allow the experiments to be repeated. Targeting the ESI procedure to the middle of exons is likely to have a bigger impact for smaller exons as the authors mention on lines 99-100. Making it clear which exon sizes for each gene were successfully targeted in this study would help give some idea of how significant a problem this might be. Perhaps Table S1 contains this information, but it was not provided. It would also help reviewers check the design strategies.
Are the experiments adequately replicated and statistical analysis adequate?
The replication and statistically analysis of the data as presented appear adequate. Figure Legends should state the statistic used to generate error bars.
Minor comments:
Specific experimental issues that are easily addressable. Are the promoters used in the vectors described universally functional? For example, is the PGK promoter functional in yeast?
Are prior studies referenced appropriately?
The manuscript may benefit from the referencing of BAC modification techniques from a wider variety of groups, such as those using CRISPR-guided recombineering (Pyne et al. 2015).
Are the text and figures clear and accurate?
The body text is very clear save minor typographical or grammatical errors. Regarding figures, some of the coloured text in Figure 1 is somewhat illegible when printed in grayscale.
Line 278 - The acronyms LAP and NLAP are not defined/explained.
Antibody section starting Line 282 may fit better next to Western Blot section.
Figure 2C - The blot images would benefit from arrows to indicate expected sizes of proteins.
Figure 3A - the graph may benefit from a dashed line at 100% to highlight that values are normalised to controls.
Figure 4 - The differences between panels B & C are unclear.
Figure 4E - The legend could provide a little more detail on cell cycle stage/status of the captured cells.
Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
Lines 23-27 are somewhat unclear and feel out of context. Perhaps the authors could clarify this as a further advantage of using BACs instead of endogenous gene modifications.
While not affecting the factual content of the paper, I would advocate that the authors format the method described in Figure S3 into a more detailed text based layout similar to that seen in a typical Nature Methods article. However, this may depend on the format required by any eventual publishing journal. That all of the work the paper was carried out in human cell lines and using human genes is a further caveat, but the authors admit this in the discussion and one would assume that most mammalian cells would respond similarly in their ability to splice out the synthetic intron.
Significance
Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
This work is a formal description of a newer method that could be useful for many of those employing bacterial artificial chromosomes in numerous studies, such as gene regulation.
Place the work in the context of the existing literature (provide references, where appropriate).
This work builds on methodology previously published by the authors - a counter-selection two-step procedure (Bird et al. 2011). It sets out to formally describe a method merely mentioned as "BAC intronization" in a later paper by some of the authors (Zheng et al. 2014). Other alternative one-step procedures are also available, but present a different set of challenges (Lyozin et al. 2014). Some newer approaches, such as those using CRISPR-guided recombineering (Pyne et al. 2015) or systems that combine CRISPR and positive/negative selection cassettes (Wang et al. 2016) may be slightly more efficient, but are also more complex in their design.
Bird et al. 2011 DOI: 10/dv776q
Pyne et al. 2015 DOI: 10/f7jx92
Wang et al. 2016 DOI: 10/f89db5
Zheng et al. 2014 DOI: 10/f5pkr6
State what audience might be interested in and influenced by the reported findings.
As a technology paper this work should have interest from a broad field of research. While the use of BACs could sometimes be considered more traditional in light of the explosion in CRISPR-based genome editing capabilities, it is definitely seeing a resurgence as the limitations of CRISPR in modifying large regions of genome become more apparent. Therefore, technologies that accelerate the modification of BACs could prove increasingly useful. As category of audience, all those involved in significant recombineering or gene/genome engineering would potentially benefit.
Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
Synthetic genomics, synthetic biology, cancer cell biology, gene and genome engineering
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