Dataset Information

Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.

ABSTRACT: The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.

SUBMITTER: Kimura Y

PROVIDER: S-EPMC4189020 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.

Kimura Yukiko Y Hisano Yu Y Kawahara Atsuo A Higashijima Shin-ichi S

Scientific reports 20141008

The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes ...[more]

PMID: 25293390

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Project description:Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (Danio rerio). Our study demonstrated that an ∼200 base-pair sequence encoding a composite tag can be efficiently "knocked-in" into the zebrafish genome using a combination of the CRISPR-Cas9 ribonucleoprotein complex and a long single-stranded DNA (lssDNA) as a donor template. Here, we targeted the sox3, sox11a, and pax6a genes to evaluate the knock-in efficiency of lssDNA donors with different structures in somatic cells of injected embryos and for their germline transmission. The structures and sequence characteristics of the lssDNA donor templates were found to be crucial to achieve a high rate of precise and heritable knock-ins. The following were our key findings: (1) lssDNA donor strand selection is important; however, strand preference and its dependency appear to vary among the target loci or their sequences. (2) The length of the 3' homology arm of the lssDNA donor affects knock-in efficiency in a site-specific manner; particularly, a shorter 50-nt arm length leads to a higher knock-in efficiency than a longer 300-nt arm for the sox3 and pax6a knock-ins. (3) Some DNA sequence characteristics of the knock-in donors and the distance between the CRISPR-Cas9 cleavage site and the tag insertion site appear to adversely affect the repair process, resulting in imprecise editing. By implementing the proposed method, we successfully obtained precisely edited sox3, sox11a, and pax6a knock-in alleles that contained a composite tag composed of FLAGx3 (or PAx3), Bio tag, and HiBiT tag (or His tag) with moderate to high germline transmission rates as high as 21%. Furthermore, the knock-in allele-specific quantitative polymerase chain reaction (qPCR) for both the 5' and 3' junctions indicated that knock-in allele frequencies were higher at the 3' side of the lssDNAs, suggesting that the lssDNA-templated knock-in was mediated by unidirectional single-strand template repair (SSTR) in zebrafish embryos.

Dataset Information

Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.

Publications

Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.

Similar Datasets

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