斑马鱼
生物
清脆的
计算生物学
基因座(遗传学)
Cas9
基因组编辑
遗传学
基因
基因组
正向遗传学
同源定向修复
DNA修复
核苷酸切除修复
作者
Haipeng Bai,Lijun Liu,Ke An,Xiaochan Lu,Michael R. Harrison,Yanqiu Zhao,Ruibin Yan,Zhi-Jie Lu,Song Li,Shuo Lin,Fang Liang,Wei Qin
出处
期刊:BMC Genomics
[Springer Nature]
日期:2020-01-21
卷期号:21 (1)
被引量:39
标识
DOI:10.1186/s12864-020-6493-4
摘要
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.
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