Boosting targeted genome editing using the hei-tag
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Evaluation Summary:
The authors describe the hei-tag, which, when added to a genome editing enzyme, results in increased editing rates in fish embryos and mammalian cell culture. The hei-tag tool could provide a valuable alternative that can potentially boost genome editing efficiency in different species and systems. The wider applicability of this approach still requires further investigation, since the improvement of editing efficiency is so far supported by experimental data on only a few targets. It would also be important to learn how the authors' design decisions affect activity, especially when benchmarked against current state-of-art genome editing tools.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straightforward purification. Immediate activity is usually granted by administration of preassembled protein/RNA complexes. We present the ‘hei-tag ( h igh e ff i ciency-tag)’ which boosts the activity of CRISPR/Cas genome editing tools already when supplied as mRNA. The addition of the hei-tag, a myc-tag coupled to an optimized NLS via a flexible linker, to Cas9 or a C-to-T (cytosine-to-thymine) base editor dramatically enhances the respective targeting efficiency. This results in an increase in bi-allelic editing, yet reduction of allele variance, indicating an immediate activity even at early developmental stages. The hei-tag boost is active in model systems ranging from fish to mammals, including tissue culture applications. The simple addition of the hei-tag allows to instantly upgrade existing and potentially highly adapted systems as well as to establish novel highly efficient tools immediately applicable at the mRNA level.
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Evaluation Summary:
The authors describe the hei-tag, which, when added to a genome editing enzyme, results in increased editing rates in fish embryos and mammalian cell culture. The hei-tag tool could provide a valuable alternative that can potentially boost genome editing efficiency in different species and systems. The wider applicability of this approach still requires further investigation, since the improvement of editing efficiency is so far supported by experimental data on only a few targets. It would also be important to learn how the authors' design decisions affect activity, especially when benchmarked against current state-of-art genome editing tools.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the …
Evaluation Summary:
The authors describe the hei-tag, which, when added to a genome editing enzyme, results in increased editing rates in fish embryos and mammalian cell culture. The hei-tag tool could provide a valuable alternative that can potentially boost genome editing efficiency in different species and systems. The wider applicability of this approach still requires further investigation, since the improvement of editing efficiency is so far supported by experimental data on only a few targets. It would also be important to learn how the authors' design decisions affect activity, especially when benchmarked against current state-of-art genome editing tools.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Improving the efficiency of genome engineering tools is a very important and competitive field. Moreover, the design of improved tools with a focus on models outside the mammalian cell editing dominated space is very welcome, as cross species activity should not be taken for granted. Thomas Thumberger et al focus on improving Cas9 activity in medaka and zebrafish embryos by rethinking about the "bells and whistles" attached to the enzyme in its commonly available variants.
They first develop an assay that links Cas9 activity to a phenotypic readout (retinal pigmentation controlled by oc2) in fish embryos. Using the first Cas9 variant reported to edit zebrafish embryos (Hwang et al 2013) as benchmark, they test an improved Cas9 variant (Zhang et al 2014) and their own heiCas9, and report up to 8-fold …
Reviewer #1 (Public Review):
Improving the efficiency of genome engineering tools is a very important and competitive field. Moreover, the design of improved tools with a focus on models outside the mammalian cell editing dominated space is very welcome, as cross species activity should not be taken for granted. Thomas Thumberger et al focus on improving Cas9 activity in medaka and zebrafish embryos by rethinking about the "bells and whistles" attached to the enzyme in its commonly available variants.
They first develop an assay that links Cas9 activity to a phenotypic readout (retinal pigmentation controlled by oc2) in fish embryos. Using the first Cas9 variant reported to edit zebrafish embryos (Hwang et al 2013) as benchmark, they test an improved Cas9 variant (Zhang et al 2014) and their own heiCas9, and report up to 8-fold "enhanced activity" in medaka and 27-fold in zebrafish, when comparing to the Hwang et al variant. Improvements over the Zhang et al variant, tested only in medaka, were more modest.
Lacking a phenotypic assay in mammalian cells, the authors use genome editing outcomes deconvolution software (ICE and TIDE) on Sanger sequencing data to compare the editing efficiency of the Hwang variant, a commercially available Cas9, and heiCas9. HeiCas9 scores better in both analyses, and also shows a clear improvement at the ICE knockout score.
Finally, the authors move away from DSB inducing methods of genome editing and construct a heiBE4 (C to T base editor) variant. By adopting their phenotypic assay in medaka to introduce a stop codon (CAG>TAG) in oc2, they show stronger pigment loss phenotypes in injected embryos when using heiBE4, compared to the original BE4. They further quantify and confirm the high rate of C>T transitions by sequencing.
Overall, the manuscript is well written and the results are clearly presented. Boosting genome editing without modifying the primary sequence of the enzyme is a very interesting approach, and has been reported before (Liu et al 2021). Such methods could also be compatible with artificially evolved Cas9 variants (e.g. high fidelity, relaxed PAM recognition) or even other Cas enzymes (e.g. Cas12), providing an orthogonal approach to increase their activity.
The authors also provide evidence that the hei-tag is not restricted to the conventional DSB inducing approach, by trying a BaseEditor.
How their height-tag works to improve genome editing is not investigated in detail. Knowing the mechanistic underpinnings can help predict the usefulness or lack thereof across different organisms, developmental stages, or cell states. Moreover, the balance between ON- and OFF-target activity is not considered, an important parameter for cell culture experiments where outcrossing is not possible to segregate non-specific modifications of the genome. As a result, the mammalian cell culture data are interesting, but don't add much to the value of the hei-tag.
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Reviewer #2 (Public Review):
The paper focuses on describing a novel tag (named hei-tag) consisting of two optimized NLS sequences and a Myc epitope separated by a linker peptide. This tag fused with Cas9 ORF increases the efficiency of genome editing in fish embryos following mRNA+sgRNA injection. The authors assess these results with a rigorous quantification of pigmentation reduction in fish embryos following the targeting of the pigmentation locus Oca2 in Medaka and zebrafish embryos. The same improved version of Cas9 is tested in mammalian cells in culture comparing the results with older version of Cas9. Finally the authors fuse the hei-tag to the base editor BE4-Gam showing also in this case an increased activity. The data would be more significant for the community if the injection of the Cas9 optimized construct would be tested …
Reviewer #2 (Public Review):
The paper focuses on describing a novel tag (named hei-tag) consisting of two optimized NLS sequences and a Myc epitope separated by a linker peptide. This tag fused with Cas9 ORF increases the efficiency of genome editing in fish embryos following mRNA+sgRNA injection. The authors assess these results with a rigorous quantification of pigmentation reduction in fish embryos following the targeting of the pigmentation locus Oca2 in Medaka and zebrafish embryos. The same improved version of Cas9 is tested in mammalian cells in culture comparing the results with older version of Cas9. Finally the authors fuse the hei-tag to the base editor BE4-Gam showing also in this case an increased activity. The data would be more significant for the community if the injection of the Cas9 optimized construct would be tested as protein as this is the most efficient and commonly used approach in fish experiments. Similarly the comparison with the BE4-Gam should be extended to the more recent family of improved Cytidine Base Editors including the ancBE4Max that was optimized for nuclear shuttling among other properties. In the present manuscript the presence of the Myc epitope in the hei-tag is not tested in any of its possible applications and it remains unclear what is the utility of this part of the hei-tag system.
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Reviewer #3 (Public Review):
In this manuscript, Thumberger et al. developed a novel high-efficiency tag to be used with existing CRISPR/Cas9 tools that boosted the editing efficiency in fish (medaka, zebrafish) and mammalian cell culture. Compared to the baseline gene editing methods chosen by the authors, hei-tag improved bi-allelic editing efficiency in medaka fish by about 30% and resulted in about 10% more indels in editing mammalian cells. The authors have also shown that hei-tag can be added to other Cas9-based techniques such as base editors to boost editing efficiency.
The authors have shown convincing evidence that hei-tag improves editing efficiency compared to the baseline methods they have chosen (JDS246-Cas9, myc-Cas9) in fish, demonstrating a boost in bi-allelic targeting efficiency of the oca2 gene in making eye pigment …
Reviewer #3 (Public Review):
In this manuscript, Thumberger et al. developed a novel high-efficiency tag to be used with existing CRISPR/Cas9 tools that boosted the editing efficiency in fish (medaka, zebrafish) and mammalian cell culture. Compared to the baseline gene editing methods chosen by the authors, hei-tag improved bi-allelic editing efficiency in medaka fish by about 30% and resulted in about 10% more indels in editing mammalian cells. The authors have also shown that hei-tag can be added to other Cas9-based techniques such as base editors to boost editing efficiency.
The authors have shown convincing evidence that hei-tag improves editing efficiency compared to the baseline methods they have chosen (JDS246-Cas9, myc-Cas9) in fish, demonstrating a boost in bi-allelic targeting efficiency of the oca2 gene in making eye pigment in medaka and zebrafish. Applications beyond gene knockout in fish have been carried out in mammalian cell culture, and also in base editing tools in medaka, implicating the potential broad application of the tool.
However, it is not clear under what scenarios hei-tag carries out significant and practical improvement compared to the state-of-art gene editing techniques. Especially concerning its original purpose of editing earlier and more efficiently in early embryos, the common strategy is to inject Cas9 protein rather than mRNA, which the authors did not account for. Even in the realm of RNA-based editing tools, it is questionable whether JDS246-Cas9, a construct originally made for mammalian gene editing, is the best baseline to compare to. Part of the ambiguity originates from a lack of systematic comparison of existing editing tools in the field, but the authors would need to ensure they are comparing to the state-of-art, and demonstrate the universality and limitation of hei-tag in practical use.
Overall, I think hei-tag would be a good addition to the exiting gene editing tools and has a potential to boost editing efficiency in many systems, although its practical improvement is yet to be solidly demonstrated. Further investigation of the impact of N-terminal tags and linker structure on Cas9 specificity and efficacy will be useful to guide future improvement of protein engineering.
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