A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes
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Abstract
Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.
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###Reviewer #2:
Kroll et al. presented a strategy to achieve biallelic knockout effects in the founder (F0) generation of zebrafish, by targeting three different loci within the same target gene, with injection of Cas9 RNP mixtures. They showed that in addition to target single genes, this method could be successfully used to create double knockouts of slc24a5 and tbx5a gene pair, or tyr and ta gene pair, in F0 embryos. Strikingly, they also demonstrated direct generation of triple gene knockouts of mitfa, mpv17 and slc45a2 in F0 larvae, which fully recapitulated the pigmentation defects of the crystal mutant. Furthermore, they provide evidence of the feasibility of their method in dissecting complex and multi-parameter behavioural traits in the biallelic F0 knockouts of trpa1b, csnk1db, scn1lab genes. Interestingly, they established a …
###Reviewer #2:
Kroll et al. presented a strategy to achieve biallelic knockout effects in the founder (F0) generation of zebrafish, by targeting three different loci within the same target gene, with injection of Cas9 RNP mixtures. They showed that in addition to target single genes, this method could be successfully used to create double knockouts of slc24a5 and tbx5a gene pair, or tyr and ta gene pair, in F0 embryos. Strikingly, they also demonstrated direct generation of triple gene knockouts of mitfa, mpv17 and slc45a2 in F0 larvae, which fully recapitulated the pigmentation defects of the crystal mutant. Furthermore, they provide evidence of the feasibility of their method in dissecting complex and multi-parameter behavioural traits in the biallelic F0 knockouts of trpa1b, csnk1db, scn1lab genes. Interestingly, they established a rapid sequencing-free method to evaluate the activity of Cas9 RNP by using headloop PCR, facilitating the selection of target sites. Finally, the authors proposed a three-step protocol for F0 knockout screens in zebrafish. The strategy described here is quite impressive, and represents evident improvements of the method published by Wu et al. (Developmental Cell, 2018), which was based on the administration of four Cas9/gRNA RNPs. Nevertheless, the manuscript could be further clarified and improved in the following aspects.
What are the essential differences in methodology of this method compared with that reported by Wu et al. in 2018 (Developmental Cell)? Or why and how the target sites could be reduced to three from four?
Several genes were tested in both work, such as slc24a5, tyr, tbx16, and tbx5a, did you use or compare the same target sites in these genes as reported by Wu et al.?
Is the dosage/amount of Cas9 or RNP used in this study different or comparable with Wu et al.? Does it account for the improvement of the method described in the study?
The authors propose to design the three target sites in distinct exon within each gene. Is it really important and/or necessary to achieve high efficient biallelic knockouts? Any evidence?
According to the section of MATERIALS AND METHODS, the synthetic gRNA was made of two components, i.e., crRNA and tracrRNA. Synthesis of gRNA as a single molecule by in vitro transcription is usually more popular and economic, is it really necessary to use crRNA and tracrRNA to achieve high efficient biallelic knockouts? Any evidence?
Could headloop PCR be used for the quantification of mutagenesis efficiency (indel-producing mutation rate) of Cas9/gRNA? How sensitive is this method? Could small indels (such as 1-bp insertion or deletion) be detected by the headloop PCR?
In addition to indels, deletions between two double strand breaks induced by two gRNAs are also important for the generation of biallelic knockouts of the target gene. The authors showed the analysis of mutations in each site (such as in Fig. 2A), is it possible to quantify the distribution and contribution of all the different deletions?
Fig. 1C and 1D: The authors compared the effects of the injection of 1, 2, 3, and 4 loci. How were the 1, 2, and 3 loci selected from the four target sites? Will each of the four loci give the same or different phenotypic ratio if tested individually? Will different combinations of 2 loci or 3 loci give the same or different phenotypic ratio? Or which combination of 2 loci or 3 loci will give the highest mutagenic effect? For example, in Fig. 1C, the 3-loci showed comparable effect with 4-loci, while the 2-loci is less effective; is it possible to find other 2-loci combinations which could show higher mutagenic efficiency than the current 2-loci, such that the effect of the new 2-loci combination is as good as the 3-loci or 4-loci combination? Conversely, in Fig. 1D, the 2-loci already showed the highest mutagenic effect, is it because of this particular 2-loci combination, or any 2-loci combination will show the same efficiency?
Figure 6: The phenotypes of scn1lab F0 knockouts are more severe than those of scn1lab-/- mutant. Any explanation?
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###Reviewer #1:
Kroll and colleagues describe an efficient strategy to reliably generate F0 zebrafish embryos with (multiple) genes knocked out using CRISPR/Cas9 RNPs. In their most dramatic and broadly applicable proof-of-principle experiment, authors demonstrate successful recapitulation of the triple mutant crystal phenotype in 9/10 F0 embryos. As the authors point out, their methodology is extremely likely to be adapted for candidate genes for traits which display a range of phenotypes among wild type embryos or larvae.
The manuscript points out a rather obvious but somehow underreported feature of NHEJ-based mutagenesis: assuming random size of indels, when 100% of DNA is mutated fewer than 50% (.67x.67) of cells in an embryo will contain frameshift mutations in both alleles. Thus, successful recapitulation of a mutant phenotype in …
###Reviewer #1:
Kroll and colleagues describe an efficient strategy to reliably generate F0 zebrafish embryos with (multiple) genes knocked out using CRISPR/Cas9 RNPs. In their most dramatic and broadly applicable proof-of-principle experiment, authors demonstrate successful recapitulation of the triple mutant crystal phenotype in 9/10 F0 embryos. As the authors point out, their methodology is extremely likely to be adapted for candidate genes for traits which display a range of phenotypes among wild type embryos or larvae.
The manuscript points out a rather obvious but somehow underreported feature of NHEJ-based mutagenesis: assuming random size of indels, when 100% of DNA is mutated fewer than 50% (.67x.67) of cells in an embryo will contain frameshift mutations in both alleles. Thus, successful recapitulation of a mutant phenotype in an F0 embryo relies on mutagenesis of an essential part of the protein (not always as straightforward as it seems), utilization of other repair pathways such as MMEJ (not always reliable), or fortuitous help from largely unknown factors which skew the distribution of indel sizes (multiple guide would RNAs need to be tested without guarantee of success). Simultaneously designing several guide RNAs against the gene and co-injecting them, as the authors propose, seems to be an excellent and straightforward strategy.
My most significant criticism is that although new to zebrafish, the described strategies - use multiple guide RNAs and headloop PCR - have been successfully deployed in other systems. Adapting these strategies to the zebrafish model system offers tremendous value, but the distinction between development of new methods and adoption of existing methodologies must be considered.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 4 of the manuscript.
###Summary:
The authors describe a new efficient strategy to reliably generate F0 zebrafish embryos with (multiple) genes knocked out using CRISPR/Cas9 RNPs. They showed that in addition to target single genes, this method could be successfully used to create double knockouts of slc24a5 and tbx5a gene pair, or tyr and ta gene pair, in F0 embryos. Strikingly, they also demonstrated direct generation of triple gene knockouts of mitfa, mpv17 and slc45a2 in F0 larvae, which fully recapitulated the pigmentation defects of the …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 4 of the manuscript.
###Summary:
The authors describe a new efficient strategy to reliably generate F0 zebrafish embryos with (multiple) genes knocked out using CRISPR/Cas9 RNPs. They showed that in addition to target single genes, this method could be successfully used to create double knockouts of slc24a5 and tbx5a gene pair, or tyr and ta gene pair, in F0 embryos. Strikingly, they also demonstrated direct generation of triple gene knockouts of mitfa, mpv17 and slc45a2 in F0 larvae, which fully recapitulated the pigmentation defects of the crystal mutant. Their methodology is extremely likely to be adapted for candidate genes for traits which display a range of phenotypes among wild type embryos or larvae.
This is a new tool for the zebrafish community. Despite the presented data on several loci, it is not clear whether and how this method is better compared to a series of prior related F0 approaches. This question is the crux of this methods manuscript.
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####This manuscript is in revision at eLife (August 17, 2020)
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