Optimization of genome editing by CRISPR ribonucleoprotein for high efficiency of germline transmission of Sox9 in zebrafish.

Primordial germ cells (PGCs) are the first germline stem cells to emerge during early embryonic development and are essential for the propagation and survival of species. Genome editing creates mutagenesis possibilities in vivo, but the generation of precise mutations in PGCs is still challenging. Here, we report an optimized approach for highly efficient genome editing via introducing biallelic variations in early embryos in zebrafish. We adopted an extended, GC-rich, and chemically modified sgRNA along with microinjection of the CRISPR ribonucleoprotein (RNP) complex into the yolk sac at the 1-cell stage. We found that genome editing of Sox9a generated a high proportion of heterozygotes with edited alleles in the F generation, indicating biallelic editing. Deep sequencing and mapping the edited cells from early embryos to future tissues revealed that the edited founder has a dominantly edited allele, sox9a M1, accounting for over 99% of alleles in the testis. Specifically, all offspring of the founder inherited the edited allele, suggesting nearly complete editing of the alleles in early germline cells. Overall, the optimization delineates biallelic editing of sox9a in early embryos and transmission of edited alleles to offspring, thus presenting a method to create a desired genetic mutation line of Sox9a avoiding lengthy traditional crossbreeding.