Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish

Abstract

The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10–40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.

Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos

“…Several recent studies suggested that MMEJ may be utilized to repair TALENor CRISPR/Cas9-induced DSBs in different animal models [56,57]. While this manuscript was under review, it was reported that precise in-frame knock-in could be achieved in zebrafish by using short homologous sequences (10-40 bp) [58], although it is unknown whether MMEJ, SSA or both were involved in that process.…”

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

“…CRISPR/Cas9 genome modification is a powerful approach for generating both targeted indel mutations by non-homologous endjoining (NHEJ) repair and more precise gene editing by the introduction of specific sequences through homology-directed repair (HDR) (Auer et al, 2014;Bassett et al, 2014;Bhattacharya et al, 2015;Blitz et al, 2013;Chen et al, 2013;Friedland et al, 2013;Guo et al, 2014;Hisano et al, 2015;Kimura et al, 2014;Kotani et al, 2015;Li et al, 2015;Nakayama et al, 2013Nakayama et al, , 2014Ran et al, 2013;Shi et al, 2015;Wang et al, 2015;Yang et al, 2014). While NHEJ-mediated indel mutations have proven to be effective in animal models including mouse, zebrafish and Xenopus (Blitz et al, 2013;Kotani et al, 2015;Nakayama et al, 2013;Wang et al, 2015;Xue et al, 2014), HDR-mediated targeted DNA insertion has proven more challenging.…”

CRISPR/Cas9-Mediated Knockin and Knockout in Zebrafish

“…In 2013, Zu et al reported the first HR gene-targeting event using TALENs and a double stranded vector containing an eGFP cassette flanked by long homology arms and a germ line transmission rate of 1.5%. More recently many other laboratories have developed various methods to generate knockin alleles by HR followed by CRISPR/Cas9-induced DSB, using as donor single stranded DNA, circular or linear plasmids with short (~40 bp) or long (800-1000 bp) homology arms (Hruscha et al 2013;Hwang et al 2013a;Irion et al 2014;Shin et al 2014;He et al 2015;Hisano et al 2015). Although these methods were proven possible, their efficiency remains variable.…”

“…Key points for its success are (1) the identification of efficient sgRNAs targeting the chromosomal site of choice, for which new prescreening methods have been developed (Carrington et al 2015;Prykhozhij et al 2016); (2) the injection of the sgRNA mix with Cas9 nuclease mRNA over purified Cas9 protein that seems to prevent the donor plasmid insertion; and (3) further screening for the identification of founders due to the error-prone nature of junction sites between the endogenous locus and the donor vector. Hisano et al (2015) addressed this last point by introducing 10-40 bp homology arms into the donor vector to trigger integration events mediated by HR repair mechanisms. In parallel, Li et al (2015) developed another approach by targeting intronic regions of the gene of interest, therefore non-HR dependent.…”