sox32 Alleles Generated from Four-guide Cas9 RNP Targeting in G0 Zebrafish Embryos

We report long-read sequencing results from the sox32 allele targeted with a multi-guide Cas9 RNP strategy. Embryos were cell-injected with Cas9 RNP programmed with 4 guides simultaneously targeting different portions of the sox32 gene. Genomic DNA was extracted from each embryo and the sox32 coding sequence and intron were amplified and sequenced. Six individual embryos were evaluated. We found that the majority of sequences were unique and most involved mutations confined to the target sites rather than larger lesions spanning two or more target sites. We also found that alleles accumulated mutations over several DNA replication cycles, which may in part explain the diversity of alleles that were generated. This study is the first to use high-throughput sequencing to evaluate of an allele of this size mutated by a multi-guide Cas9 RNP strategy and provides insights into the dynamics of CRISPR-Cas9 RNP gene editing in vivo. Amplicon sequencing of 6 biological replicates using PacBio Sequencing