An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing

Abstract

Homology-directed recombination (HDR)-mediated genome editing is a powerful approach for both basic functional study and disease modeling. Although some studies have reported HDR-mediated precise editing in nonrodent models, the efficiency of establishing pure mutant animal lines that carry specific amino acid substitutions remains low. Furthermore, because the efficiency of nonhomologous end joining (NHEJ)-induced insertion and deletion (indel) mutations is normally much higher than that of HDR-induced point mutations, it is often difficult to identify the latter in the background of indel mutations. Using zebrafish as the model organism and Y box-binding protein 1 (Ybx1/) as the model molecule, we have established an efficient platform for precise CRISPR/Cas9-mediated gene editing in somatic cells, yielding an efficiency of up to 74% embryos. Moreover, we established a procedure for screening germline transmission of point mutations out of indel mutations even when germline transmission efficiency was low (<2%). To further improve germline transmission of HDR-induced point mutations, we optimized several key factors that may affect HDR efficiency, including the type of DNA donor, suppression of NHEJ, stimulation of HDR pathways, and use of Cas9 protein instead of mRNA. The optimized combination of these factors significantly increased the efficiency of germline transmission of point mutation up to 25%. In summary, we have developed an efficient procedure for creating point mutations and differentiating mutant individuals from those carrying knockouts of entire genes.

Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

“…Levels of mosaicism in gene targeting using HMEJ in zebrafish vary greatly in the reported literature (Hisano et al 2015) (Wierson et al 2018). Consistent with the observation that SAMR in noto:RFP-DR 48 is mosaic, it was recently reported that translation of Cas9 mRNA and subsequent gene editing after one-cell stage injection is not complete until the 16 or 32 cell stage while RNP injections result in appreciable nuclease activity by the 2-4 cell stage (Zhang, Zhang, and Ge 2018). Thus, noto:RFP-DR 48 is an assay system where injection conditions can be optimized to enhance somatic gene targeting and decrease mosaicism in cell types that arise from the mesoderm.…”

CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish

“…Another strategy is to use small molecules to improve HDR efficiency by suppressing the activity of the NHEJ pathway or boosting the activity of the HDR pathway. Recently, the Ge group reported that using Cas9 protein instead of mRNA, suppression of NHEJ with SCR7 and stimulation of HDR pathways with RS-1 in combination could increase the efficiency of germline transmission of point mutations up to 25% in zebrafish [24]. However, these chemical treatments influence the endogenous DNA repair processes and may be toxic during embryonic development as a result [25].…”

Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon

“…An important aspect hindering a direct comparison with several previous studies is the different methods used to evaluate the HDR efficiency, such as PCR-or restriction-assays, sequencing of a limited number of clones, genotyping based on high-resolution melting analysis, fluorescence etc. [37][38][39][40]42,44,46 . Only three studies in zebrafish have used NGS, showing lower perfect repair rates of 1-4% 41 , 1.7-3.5% 45 and <1% 43 .…”

“…With the exception of very low efficiency KI of a gene encoding red fluorescent protein in rohu carp 34 , such advanced and fine-tuned genome editing has not been developed for farmed fish, and it may be useful to learn from protocols already established in model fish species. While only a few studies have reported HDR in medaka 35,36 , several KI-strategies have been reported in zebrafish using either donor plasmids [37][38][39] or single-stranded oligodeoxynucleotides (ssODNs) [40][41][42][43][44][45] , or both 46 . Knowledge from other fish studies are somewhat inconclusive when it comes to deciding the strategy for applying HDR in salmon, as there is a lack of consensus regarding the impact of different repair templates, homology arm length and strand complementarity in the above-mentioned studies.…”