CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish
Haipeng Bai, Lijun Liu, Ke An, Xiaochan Lu, Michael R. Harrison, Yanqiu Zhao, Ruibin Yan, Zhijie Lu, Song Li, Shuo Lin, Fang Ting Liang, Wei Qin
Abstract
Abstract Background Gene targeting by homology-directed repair (HDR) can precisely edit the genome and is a versatile tool for biomedical research. However, the efficiency of HDR-based modification is still low in many model organisms including zebrafish. Recently, long single-stranded DNA (lssDNA) molecules have been developed as efficient alternative donor templates to mediate HDR for the generation of conditional mouse alleles. Here we report a method, zLOST (zebrafish long single-stranded DNA template), which utilises HDR with a long single-stranded DNA template to produce more efficient and precise mutations in zebrafish. Results The efficiency of knock-ins was assessed by phenotypic rescue at the tyrosinase ( tyr ) locus and confirmed by sequencing. zLOST was found to be a successful optimised rescue strategy: using zLOST containing a tyr repair site, we restored pigmentation in at least one melanocyte in close to 98% of albino tyr 25del/25del embryos, although more than half of the larvae had only a small number of pigmented cells. Sequence analysis showed that there was precise HDR dependent repair of the tyr locus in these rescued pigmented embryos. Furthermore, quantification of zLOST knock-in efficiency at the rps14 , nop56 and th loci by next generation sequencing demonstrated that zLOST showed a clear improvement. We utilised the HDR efficiency of zLOST to precisely model specific human disease mutations in zebrafish with ease. Finally, we determined that this method can achieve a germline transmission rate of up to 31.8%. Conclusions In summary, these results show that zLOST is a useful method of zebrafish genome editing, particularly for generating desired mutations by targeted DNA knock-in through HDR.