Homology-Directed Repair by CRISPR–Cas9 Mutagenesis inXenopusUsing Long Single-Stranded Donor DNA Templates via Simple Microinjection of Embryos

Abstract

We describe a step-by-step procedure to perform homology-directed repair (HDR)-mediated precise gene editing inXenopusembryos using long single-stranded DNA (lssDNA) as a donor template for HDR in conjunction with the CRISPR–Cas9 system. A key advantage of this method is that it relies on simple microinjection of fertilizedXenopuseggs, resulting in high yield of healthy founder embryos. These embryos are screened for those animals carrying the precisely mutated locus to then generate homozygous and/or heterozygous mutant lines in the F1generation. Therefore, we can avoid the more challenging “oocyte host transfer” technique, which is particularly difficult forXenopus tropicalis, that is required for an alternate HDR approach. Several key points of this protocol are (1) to use efficiently active single-guide RNAs for targeting, (2) to use properly designed lssDNAs, and (3) to use 5′-end phosphorothioate-modification to obtain higher-efficiency HDR.

Electroporation-based Easi-CRISPR yields biallelic insertions of EGFP-HiBiT cassette in immortalized chicken oviduct epithelial cells

“…Here, even if the transgene cassette was 1.1 kb in size, much longer than the ssODN used in the 2 aforementioned studies, irrespective of Cas9/sgRNA delivery, a similar result was obtained—the KI efficiency for the lssDNA with shorter HAs (80 bp each) was considerably higher than that for the circular (plasmid) and linearized (PCR fragment) transgene cassettes with longer HAs (600 bp each) ( Figure 5 ). This is also in line with the findings in chicken PGC cells ( Idoko-Akoh et al, 2018 ), Xenopus eggs ( Nakayama et al, 2022 ), and mouse embryos ( Codner et al, 2018 ; Miura et al, 2018 ; Bennett et al, 2021 ).…”

Modelling human genetic disorders in Xenopus tropicalis

“…Whilst this is straightforward for LOF frameshift, deletion, exon skipping and nonsense mutations, single-base changes, however, are more of a challenge and require the improvement of base-editing techniques in the frog ( Shi et al, 2019 ; Park et al, 2017 ; Naert et al, 2021a , b ). Heterozygous, non-mosaic, precise changes, including small insertions, have been achieved by engineering the oocyte genome ( Aslan et al, 2017 ; Martin, 2023 ) or by injecting long single-stranded DNA templates for insertion into the frog genome via endogenous homology-directed repair ( Nakayama et al, 2020 , 2022c ).These methodologies remain inefficient yet worthwhile.…”