Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System

Armstrong, Gary A. B.; Liao, Meijiang; You, Zhipeng; Lissouba, Alexandra; Chen, Brian Edwin; Drapeau, Pierre

doi:10.1371/journal.pone.0150188

Cited by 109 publications

(87 citation statements)

References 16 publications

(19 reference statements)

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“…In recent studies, HDR has been used to generate knockin lines of zebrafish with various efficiencies [12][13][14][24][25][26][27] . For most of reported zebrafish lines generated via HDR, single-stranded oligodeoxynucleotide (ssODN) was usually used as a repair template 14,[24][25][26][27] . This strategy can only achieve genomic alteration with a few of bps such as point mutation and short tag insertion.…”

Section: Discussionmentioning

confidence: 99%

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Efficient replacement of long DNA fragments via non-homologous end joining at non-doding regions

Cao

et al. 2020

Preprint

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Genomic DNA replacement for achieving sophisticated genetic manipulation is implemented currently through homogenous recombination/homology-dependent repair (HR/HDR). Here we report an efficient DNA fragment replacement method that is mediated by non-homologous end joining (NHEJ)-dependent DNA repair at two sites of CRISPR/Cas9-induced double-strand breaks at non-coding genomic regions flanking the exons of targeted genes. We demonstrated this method by generating three conditional alleles and two reporter lines of zebrafish. Functional assays of the conditional alleles proved that the genomic sequence between two inserted loxP sites was deleted by the Cre recombinase, and the phenotype after Cre-induced excision was comparable to previously reported mutants or morphants. Furthermore, combining double-fluorescence expression donor vectors, we showed that the efficiency of this NHEJ-mediated DNA replacement was around 3 times larger than that of HR/HDR-mediated approach. Our method provides a feasible strategy for genomic DNA replacement in zebrafish, which can be applicable for other organisms as well.

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Section: Discussionmentioning

confidence: 99%

“…However, the requirement for pre-screening cannot be met in many cases. Without pre-screening, genomic editing mediated by HDR in zebrafish shows relatively low efficiencies [25][26][27] , in particular for long DNA fragments 12,14 . This makes it necessary to develop more efficient methods for achieving long DNA fragments replacement.…”

Section: Discussionmentioning

confidence: 99%

Efficient replacement of long DNA fragments via non-homologous end joining at non-doding regions

Cao

et al. 2020

Preprint

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“…Knock-in of specific mutations and exogenous DNA sequences has been achieved in zebrafish through both homology-independent and homology-directed repair (HDR),61–64 but this approach is currently less efficient than generation of LOF alleles 61…”

Section: Generating Zebrafish Models Of Rare Genetic Diseasesmentioning

confidence: 99%

Use of zebrafish models to investigate rare human disease

2018

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Rare diseases are collectively common and often extremely debilitating. Following the emergence of next-generation sequencing (NGS) technologies, the variants underpinning rare genetic disorders are being unearthed at an accelerating rate. However, many rare conditions lack effective treatments due to their poorly understood pathophysiology. There is therefore a growing demand for the development of novel experimental models of rare genetic diseases, so that potentially causative variants can be validated, pathogenic mechanisms can be investigated and therapeutic targets can be identified. Animal models of rare diseases need to be genetically and physiologically similar to humans, and well-suited to large-scale experimental manipulation, considering the vast number of novel variants that are being identified through NGS. The zebrafish has emerged as a popular model system for investigating these variants, combining conserved vertebrate characteristics with a capacity for large-scale phenotypic and therapeutic screening. In this review, we aim to highlight the unique advantages of the zebrafish over other in vivo model systems for the large-scale study of rare genetic variants. We will also consider the generation of zebrafish disease models from a practical standpoint, by discussing how genome editing technologies, particularly the recently developed clustered regularly interspaced repeat (CRISPR)/CRISPR-associated protein 9 system, can be used to model rare pathogenic variants in zebrafish. Finally, we will review examples in the literature where zebrafish models have played a pivotal role in confirming variant causality and revealing the underlying mechanisms of rare diseases, often with wider implications for our understanding of human biology.

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“…Several studies have used ssODNs with homology arms between 20 and 50 bp to incorporate small changes at the CRISPR-Cas9 induced DSBs. Most integrations are imprecise [12,25,30,34,35] suggesting that the mechanism may not be HR as seen in ES cells. In many cases, precise integration is not necessary, such as intronic insertion of loxP sites or exonic insertion of a cassette containing stop codons in all frames [12,25].…”

Section: Targeted Mutagenesis In Zebrafishmentioning

confidence: 99%

Zebrafish Genome Engineering Using the CRISPR–Cas9 System

et al. 2016

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Geneticists have long sought the ability to manipulate vertebrate genomes by directly altering the information encoded in specific genes. The recently discovered CRISPR-Cas9 endonuclease has the ability to bind single loci within vertebrate genomes and generate double-strand breaks at those sites. These double stranded breaks induce an endogenous double-strand break repair response that results in small insertions or deletions at the targeted site. Alternatively, a template can be supplied in which case homology-directed repair results in generation of engineered alleles at the break site. These changes alter the function of the targeted gene facilitating the analysis of gene function. This tool has been widely adopted in the zebrafish model; we discuss the development of this system in the zebrafish and how it can be manipulated to facilitate genome engineering.

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Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System

Cited by 109 publications

References 16 publications

Efficient replacement of long DNA fragments via non-homologous end joining at non-doding regions

Efficient replacement of long DNA fragments via non-homologous end joining at non-doding regions

Use of zebrafish models to investigate rare human disease

Zebrafish Genome Engineering Using the CRISPR–Cas9 System

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