Abstract
Introducing desired mutations into the genome of model organisms is a priority for all research focusing on protein function and disease modeling. The need to create stable mutant lines has resulted in the rapid advancement of genetic techniques over the last few decades from chemical mutagenesis and zinc finger nucleases to clustered regularly interspaced short palindromic repeats (CRISPR) and homology-directed repair (HDR). However, achieving consistently high success rates for direct mutagenesis in zebrafish remains one of the most sought-after techniques in the field. Several genes have been modified using HDR in zebrafish, but published success rates range widely, suggesting that an optimal protocol is required. In this review, we compare target genes, techniques, and protocols from 50 genes that were successfully modified in zebrafish using HDR to find the statistically best variables for efficient HDR rates.
Highlights
Introducing specific mutations into the genome of model organisms is a long-standing goal for many researchers
Other genes repaired with 300 nucleotide long single-stranded DNA (lssDNA) templates demonstrated an impressive rate of homology-directed repair (HDR) (11– 40%) over other template sizes, these rates were lower than the 98.5% achieved while modifying tyr (Bai et al, 2020)
When RS-1 was used in combination with an nonhomologous end joining (NHEJ) inhibitor, such as SCR7 or Nu7441, the rate of homologous repair increased significantly to 74% (Zhang et al, 2018)
Summary
Introducing specific mutations into the genome of model organisms is a long-standing goal for many researchers. The variations in HDR protocols include the type of template, length of the homology arms, symmetry of repair template, choice of endonuclease, endonuclease mRNA or protein, targeted strand, injection into the yolk or cell, the introduction of nonhomologous end joining (NHEJ)-inhibiting or HDR-enhancing drugs, injection or incubation with inhibitors With all these variations, we asked: Which factors contribute to the highest success rate?. Comparison of the most successfully modified genes (somatic and germline modified), flna, tyr, noto, tbx, rb1, msna, twist, rpl, krtt1c19e, anxa2a, and ybx (with use of drug), suggests there is an advantage to injecting into the cell vs the yolk (Hisano et al, 2015; Moreno-Mateos et al, 2017; Burg et al, 2018; Zhang et al, 2018; Bai et al, 2020; Wierson et al, 2020). While the number of studies with HDR-related drugs is limited, the current results suggest that combinations of small interfering molecules could target core repair proteins in the majority of end joining processes
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