|ZFIN ID: ZDB-PUB-200123-3|
CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish
Bai, H., Liu, L., An, K., Lu, X., Harrison, M., Zhao, Y., Yan, R., Lu, Z., Li, S., Lin, S., Liang, F., Qin, W.
|Source:||BMC Genomics 21: 67 (Journal)|
|Registered Authors:||Harrison, Michael, Lin, Shuo, Qing, Wei|
|Keywords:||CRISPR/Cas9, Disease modeling, Genome editing, Homology-directed repair, Long single-stranded DNA, Next-generation sequencing, Zebrafish|
|PubMed:||31964350 Full text @ BMC Genomics|
Bai, H., Liu, L., An, K., Lu, X., Harrison, M., Zhao, Y., Yan, R., Lu, Z., Li, S., Lin, S., Liang, F., Qin, W. (2020) CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish. BMC Genomics. 21:67.
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 tyr25del/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.