CrispRVariants charts the mutation spectrum of genome engineering experiments

Lindsay, Helen; Burger, Alexa; B, Biyong; Felker, Anastasia; Hess, Christopher; Zaugg, Jonas; Chiavacci, Elena; Anders, Carolin; Jinek, Martin; Mosimann, Christian; Robinson, Mark D.

doi:10.1038/nbt.3628

Cited by 156 publications

(174 citation statements)

References 22 publications

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“…mpv17 crispants showed no apparent other phenotype than iridophore loss and did not have a significantly higher mortality than uninjected siblings at 5 dpf. Sanger sequencing from PCR-isolated clones of the sgRNA target region in injected individuals and allele-level analysis with CrispRVariants (Burger et al, 2016; Lindsay et al, 2016) revealed exceedingly high mutagenesis efficiency, with frameshift deletions as most predominant mutation event that is predicted to result in early termination of translation (Supplemental Figure 1). These results corroborate that direct disruption of the mpv17 ORF results in iridophore defects.…”

Section: Resultsmentioning

confidence: 99%

A defect in the mitochondrial protein Mpv17 underlies the transparent casper zebrafish

D'Agati

Beltre

Sessa

et al. 2017

Developmental Biology

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The casper strain of zebrafish is widely used in studies ranging from cancer to neuroscience. casper offers the advantage of relative transparency throughout adulthood, making it particularly useful for in vivo imaging by epifluorescence, confocal, and light sheet microscopy. casper was developed by selective breeding of two previously described recessive pigment mutants: 1) nacre, which harbors an inactivating mutation of the mitfa gene, rendering the fish devoid of pigmented melanocytes; and 2) roy orbison, a mutant with so-far unidentified genetic cause that lacks reflective iridophores. To clarify the molecular nature of the roy orbison mutation, such that it can inform studies using casper, we undertook an effort to positionally clone the roy orbison mutation. We find that roy orbison is caused by an intronic defect in the gene mpv17, encoding an inner mitochondrial membrane protein that has been implicated in human mitochondrial DNA depletion syndrome. The roy orbison mutation is phenotypically and molecularly remarkably similar to another zebrafish iridophore mutant called transparent. Using Cas9-induced crispants and germline mutants with disrupted mpv17 open reading frame, we show in trans-heterozygote embryos that new frameshift alleles of mpv17, roy orbison, and transparent fail to complement each other. Our work provides genetic evidence that both roy orbison and transparent affect the mpv17 locus by a similar if not identical genetic lesion. Identification of mpv17 mutants will allow for further work probing the relationship between mitochondrial function and pigmentation, which has to date received little attention.

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Section: Resultsmentioning

confidence: 99%

A defect in the mitochondrial protein Mpv17 underlies the transparent casper zebrafish

D'Agati

Beltre

Sessa

et al. 2017

Developmental Biology

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“…In RGB embryos, dsRED2 labels endothelial, hematopoietic, and endocardial progenitors (lmo2) in red [14], EGFP marks all lateral plate mesoderm lineages (drl) including pectoral fins in green [15], and AmCyan reveals the differentiated cardiomyocytes (myl7 ) in blue [16]; consequently, RGB enables in vivo imaging of all cardiovascular and additional LPM lineages over the first 3 days of development. From F0 outcrosses that transmitted mutant tbx5a alleles, we genotyped adult F1 zebrafish for the presence of mutated tbx5a alleles by tail clipping, PCR, sequencing, and CrispRVariants analysis [17]. From the recovered germline alleles, we kept heterozygous strains for the lesions c.21_25del and c.22_31del (hence forward abbreviated as tbx5a Δ5 and tbx5a Δ10 ) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Early frameshift alleles of zebrafish <em>tbx5a</em> that fail to develop the heartstrings phenotype

Chiavacci¹,

Kirchgeorg²,

Felker³

et al. 2017

Matters

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“…CrispRVariants is another tool that offers functionality to analyze deep-sequencing data by quantifying mosaicism and allele-specific gene editing, as well as multisequence alignment views 60 .…”

Section: Introductionmentioning

confidence: 99%

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments

et al. 2018

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CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNARNARNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4–5 weeks by non-experts, as well as computational data analysis that can be performed in 1–2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.

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CrispRVariants charts the mutation spectrum of genome engineering experiments

Cited by 156 publications

References 22 publications

A defect in the mitochondrial protein Mpv17 underlies the transparent casper zebrafish

A defect in the mitochondrial protein Mpv17 underlies the transparent casper zebrafish

Early frameshift alleles of zebrafish <em>tbx5a</em> that fail to develop the heartstrings phenotype

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments

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