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

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 singlestranded 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.

Synthesis of DNA Origami Scaffolds: Current and Emerging Strategies

“…Originally, due to the complexity of long-ssDNA-strand synthesis and the limitations of chemical synthesis [ 134 ], DNA was only utilized for short insertions (100–200 bases), while dsDNA donors were utilized for insertions of lengths greater than 100 bases [ 135 , 136 ]. However, recent studies have shown that ssDNA donor templates could have higher efficiency for HDR compared to dsDNA donor templates [ 137 , 138 ]. ssDNA donor templates are now being explored by multiple groups for genome editing.…”

Homology-Directed Repair in Zebrafish: Witchcraft and Wizardry?

“…Germline transmission success rates from injecting into the yolk range from 12.5% (Burg et al, 2016) to 37.5% (Burg et al, 2018), although this range results from a limited number of studies. Comparison of the most successfully modified genes (somatic and germline modified), flna, tyr, noto, tbx20, rb1, msna, twist2, rpl18, krtt1c19e, anxa2a, and ybx1 (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). Comparing the techniques used for these 11 genes and 14 modifications (tyr was modified by four different labs), 12 out of 14 techniques utilized injecting into the cytoplasm of the 1-or 2-cell stage embryo.…”

“…The use of Cas9 mRNA or Cas9 protein varies within the field. The highest success rates for germline transmission of HDR modifications utilized Cas9 mRNA and long single-stranded oligonucleotides or double-stranded templates (Bai et al, 2020;Wierson et al, 2020); however, no comparisons with Cas9 protein have been made (Hisano et al, 2015). A 2018 study compared HDR success using Cas9 mRNA vs. Cas9 protein on the ybx1 gene (Zhang et al, 2018).…”

“…This review will discuss the HDR protocols currently used for the modification of the zebrafish genome. The majority of zebrafish studies agree: only sgRNAs with high cutting efficiencies (>60%) should be used, chemical modification of the repair template was not present, the repair template must overlap the double-strand break (DSB) site (asymmetrically or symmetrically), microinjections occur during the 1-2 cell stage, the protospacer adjacent motif (PAM) site must be altered to prevent cutting of successfully repaired targets, and the DSB cut site should be within 20 nucleotides of the target nucleotide (Paquet et al, 2016;Boel et al, 2018;Burg et al, 2018;Bai et al, 2020). There are few exceptions to the target/cut site proximity standard where cut sites are a significant distance away from their target loci (Moreno-Mateos et al, 2017;Luo et al, 2018;Eschstruth et al, 2020).…”

Prediction of off-target effects of the CRISPR/Cas9 system for design of sgRNA

“…However, this method has not been perfected as the limitation of 20 nucleotides in the sgRNA exposes this method to the possibility of being off-target [31][32][33][34] . Since it only contains 20 nucleotides, the same sequence is vulnerable to repetition within the genome and therefore the Cas9 enzyme has the possibility of being guided to the wrong locus, which in turn leads to mutations [35][36][37][38][39] .…”