Rapid and efficient cataract gene evaluation in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes

Zhao, Duran; Jones, Johanna L; Gasperini, Robert; Charlesworth, Jac; Liu, Guei‐Sheung; Burdon, Kathryn P.

doi:10.1016/j.ymeth.2020.12.004

Cited by 17 publications

(13 citation statements)

References 34 publications

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“…Because the ScCas9 and SpCas9 sequences are homologous and a previous result showed that sgRNA scaffold substitution did not affect the efficiency (Figure 2), we presumed that identical modifications from SpCas9 can be used to optimize the ScCas9 system [23,25,26]. Both the ScCas9 protein and chemically modified crRNAs increased the indel frequency similar to SpCas9.…”

Section: Discussionmentioning

confidence: 77%

“…Injection of the same amount of ScCas9 mRNA into the one-cell stage zebrafish embryos worked but greatly decreased the percentage of embryos displaying pigment loss compared with the SpCas9 mRNA group (Figure 1C). A previous study demonstrated that the SpCas9 protein can increase the indel frequency compared with SpCas9 mRNA in zebrafish [23]. Therefore, we purified recombinant ScCas9 protein using Escherichia coli expression system (Figure 1A and Figure S1).…”

Section: Sccas9 Rnp Complexes Provide Robust Genome Editing In Zebrafishmentioning

confidence: 99%

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Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM

Liu

Liang

Dong

et al. 2021

Cells

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The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5′-NNG-3′ PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.

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Section: Discussionmentioning

confidence: 77%

Section: Sccas9 Rnp Complexes Provide Robust Genome Editing In Zebrafishmentioning

confidence: 99%

Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM

Liu

Liang

Dong

et al. 2021

Cells

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“…While dgRNP-based mutagenesis utilizing chemically synthesized crRNAs have been shown to efficiently disrupt gene function in F0 zebrafish even as single dgRNP injections [31][32][33][34][35][36][37], it is becoming clear that the efficacy of target gene disruption depends on the mutagenic ability of each designed crRNA, as also suggested by the recent report showing variable efficiency of synthetic crRNAs in gene editing [37]. As such, injection of single dgRNPs per gene can lead to varied mutagenesis efficiency, resulting in variable and incosistent phenotypic observations across crRNAs targeted to the same gene in injected F0 animals.…”

Section: Injections Of Single Vs Triple Dgrnps Per Genementioning

confidence: 99%

“…While dgRNP-based mutagenesis has been shown to efficiently disrupt gene function in F0 zebrafish even as single dgRNP injections [23][24][25][26][27][28][29], it is becoming clear that anticipated phenotypic detections can depend on target genes and the mutagenic ability of each designed crRNA. This is likely due to varied genetic compensatory responses to mutations in individual loci [12][13][14]23] and the variable efficiency of synthetic crRNAs in gene editing [24,29].…”

Section: Injections Of Single and Dual Vs Triple Dgrnps Per Genementioning

confidence: 99%

“…Many studies have reported efficient methods for generating F0 zebrafish mutants using in vitrotranscribed guide RNA (gRNA) [16][17][18][19][20][21][22], or more recently, using a dual-guide CRISPR/Cas9 ribonucleoprotein (dgRNP) system that utilizes a chemically synthesized duplex of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) [23][24][25][26][27][28][29][30]. However, there is currently no integrated, standardized approach that is well established in the community, making it difficult to select which method to utilize based on pre-existing and emerging information in the rapidly evolving field.…”

Section: Introductionmentioning

confidence: 99%

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Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish

Quick

Parab

Tolbert

et al. 2021

Preprint

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The zebrafish is a valuable vertebrate model to study cardiovascular formation and function due to the facile visualization and rapid development of the circulatory system in its externally growing embryos. Despite having distinct advantages, zebrafish have paralogs of many important genes, making reverse genetics approaches inefficient since generating animals bearing multiple gene mutations requires substantial efforts. Here, we present a simple and robust synthetic CRISPR RNA/Cas9-based mutagenesis approach for generating biallelic F0 zebrafish knockouts. Using a dual-guide synthetic CRISPR RNA/Cas9 ribonucleoprotein (dgRNP) system, we show that simultaneous injections of three distinct dgRNPs per gene into one-cell stage embryos dramatically enhanced the efficiency of biallelic gene disruptions with greater consistency than any single dgRNP injections tested on 4 different genes. Importantly, this mutagenesis method is capable of disrupting genes encoding distinct types of proteins, including secreted and membrane-bound forms, and the generated F0 animals fully phenocopied the endothelial and peri-vascular defects observed in corresponding stable mutant homozygotes. Moreover, we provide evidence that this approach faithfully recapitulated the trunk vessel phenotypes resulting from the genetic interaction between two vegfr2 zebrafish paralogs. Therefore, our approach offers a highly efficient genetic platform to quickly assess novel and redundant gene function in F0 zebrafish.

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