Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction
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Evaluation Summary:
This is an important study that comprehensively compares the activities of different base editors in both medaka and zebrafish. The authors also provide a web tool for experimental design allowing approximately 30% of known human disease associated nucleotide variants to be modeled in fish with validated editors within days following injection. While other studies have shown similar activities in zebrafish, the authors nicely demonstrate the ability to generate phenotypes using different base editors in both zebrafish and medaka that correlate with specific base changes. This gene editing system coupled with the ability to design gRNAs efficiently with a web interface will likely have a lasting impact on the field.
(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. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka ( Oryzias latipes ) and zebrafish ( Danio rerio ), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3 , ube2b , usp44 , and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.
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Evaluation Summary:
This is an important study that comprehensively compares the activities of different base editors in both medaka and zebrafish. The authors also provide a web tool for experimental design allowing approximately 30% of known human disease associated nucleotide variants to be modeled in fish with validated editors within days following injection. While other studies have shown similar activities in zebrafish, the authors nicely demonstrate the ability to generate phenotypes using different base editors in both zebrafish and medaka that correlate with specific base changes. This gene editing system coupled with the ability to design gRNAs efficiently with a web interface will likely have a lasting impact on the field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the …
Evaluation Summary:
This is an important study that comprehensively compares the activities of different base editors in both medaka and zebrafish. The authors also provide a web tool for experimental design allowing approximately 30% of known human disease associated nucleotide variants to be modeled in fish with validated editors within days following injection. While other studies have shown similar activities in zebrafish, the authors nicely demonstrate the ability to generate phenotypes using different base editors in both zebrafish and medaka that correlate with specific base changes. This gene editing system coupled with the ability to design gRNAs efficiently with a web interface will likely have a lasting impact on the field.
(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. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this paper, the authors explore the potential use of next generation CRISPR base editors in zebrafish and medaka embryos. They conduct extensive testing in F0 (injected) animals on existing loci and show impressive and overall precise gene editing that can be bi-allelic. The outcomes are provocative, and the summary figure makes a strong case on the overall potential for this approach to help understand the genetic basis underlying vertebrate genomic sequence changes such as human VUSes.
This well-presented work is balanced by some key technical questions.
First, base editors are well-described to cause double-stranded DNA breaks as an unintended consequence of their obligate 'nickase' enzymatic function. Prior work in the rapidly dividing zebrafish embryo has shown that single-stranded DNA nicks nearly …
Reviewer #1 (Public Review):
In this paper, the authors explore the potential use of next generation CRISPR base editors in zebrafish and medaka embryos. They conduct extensive testing in F0 (injected) animals on existing loci and show impressive and overall precise gene editing that can be bi-allelic. The outcomes are provocative, and the summary figure makes a strong case on the overall potential for this approach to help understand the genetic basis underlying vertebrate genomic sequence changes such as human VUSes.
This well-presented work is balanced by some key technical questions.
First, base editors are well-described to cause double-stranded DNA breaks as an unintended consequence of their obligate 'nickase' enzymatic function. Prior work in the rapidly dividing zebrafish embryo has shown that single-stranded DNA nicks nearly immediately become DS DNA breaks during the rapid DNA replication phase. The authors do not address this issue at all, and instead suggest there are no DS DNA break off-target effects.
Second, in addition to the potential for DS DNA break off-target effects, there could be unanticipated other pathways being activated in F0 'editants' confounding their analyses. This is well-described for a number of genomic engineering tools, from RNAi, siRNA, shRNA, 'crispants' and 'morphants'. The presence of at least one such pathway was readily detected using differential transcriptomic work. I do not see any such approach in this manuscript.
Finally, the reason to ask any team to go through germline is to explore the area of 'what we do not know we do not know.' We've had false starts (i.e. from early 'crispant' work) where the promise that F1-incross data (F2 animals) simply do not reflect what was seen in the F0 work.
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Reviewer #2 (Public Review):
The manuscript describe a novel and user friendly on live tool to design sgRNA oligos to use Base editors in zebrafish and Medaka and easily adaptable to other model organisms. The experimental part offers some examples of the validity of their approach using in both fish species various state-of -the-art base editors including some never tested before in fish. They demonstrate that high efficient and specific base conversions can be achieved in F0 injected embryos allowing the rapid assessment of the phenotype linked to the mutation. Examples with both Adenine and Citosine base editors are presented allowing to phenocopy known mutant phenotypes as well as to test missense mutations in novel candidate genes for congenital heart disease. Overall this work nicely illustrate the power of bese editors as highly …
Reviewer #2 (Public Review):
The manuscript describe a novel and user friendly on live tool to design sgRNA oligos to use Base editors in zebrafish and Medaka and easily adaptable to other model organisms. The experimental part offers some examples of the validity of their approach using in both fish species various state-of -the-art base editors including some never tested before in fish. They demonstrate that high efficient and specific base conversions can be achieved in F0 injected embryos allowing the rapid assessment of the phenotype linked to the mutation. Examples with both Adenine and Citosine base editors are presented allowing to phenocopy known mutant phenotypes as well as to test missense mutations in novel candidate genes for congenital heart disease. Overall this work nicely illustrate the power of bese editors as highly efficient and specific tools to generate precise point mutations alleles in fish. It illustrates the power of this approach for human disease modelling in these animal models supporting their work flow approach with a set of nice examples that allow the authors to experimentally define the optimal editing window for the different base editors.
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Reviewer #3 (Public Review):
This is an important study that comprehensively compares the activities of different base editors in both medaka and zebrafish. The authors also provide a web tool for experimental design allowing approximately 30% of known human disease associated nucleotide variants to be modeled in fish with validated editors within days following injection. While other studies have shown similar activities in zebrafish, the authors nicely demonstrate the ability to generate phenotypes using different base editors in both zebrafish and medaka that correlate with specific base changes. The manuscript is nicely put together, and the data presented support their conclusions.
Some of the most impressive data presented clearly demonstrates a high degree of precise mutagenesis in the F0 generation and the ability to generate …
Reviewer #3 (Public Review):
This is an important study that comprehensively compares the activities of different base editors in both medaka and zebrafish. The authors also provide a web tool for experimental design allowing approximately 30% of known human disease associated nucleotide variants to be modeled in fish with validated editors within days following injection. While other studies have shown similar activities in zebrafish, the authors nicely demonstrate the ability to generate phenotypes using different base editors in both zebrafish and medaka that correlate with specific base changes. The manuscript is nicely put together, and the data presented support their conclusions.
Some of the most impressive data presented clearly demonstrates a high degree of precise mutagenesis in the F0 generation and the ability to generate specific phenotypes. This will greatly enhance the ability to test whether single nucleotide variants from human disease association studies have an impact on health. This coupled with the ability to design gRNAs efficiently with a web interface will likely have a lasting impact on the field.
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