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.
Date: 2020
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
MeSH Terms:
  • Amino Acid Substitution
  • Animals
  • CRISPR-Cas Systems*
  • DNA, Single-Stranded*
  • Gene Editing*
  • Gene Knock-In Techniques
  • Gene Targeting
  • Genetic Predisposition to Disease
  • Humans
  • Loss of Function Mutation
  • RNA, Guide
  • Sequence Analysis, DNA
  • Zebrafish/genetics*
PubMed: 31964350 Full text @ BMC Genomics
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.
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%.
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.