Highly efficient genome editing using RNA-guided base editors in mouse embryos

Abstract

The programmable nuclease, especially, CRISPR (clustered regularly interspaced short palindromic repeat) - Cas9 (CRISPR associated protein 9) has been significantly concerned as a powerful genome editing tool. The CRISPR-Cas9 system was dramatically developed and widely used to study gene functions and to generate model organisms in various animals and plants because it is highly specific and convenient to use. The applications were developed not only inducing double strand breaks (DSBs) effectively and precisely but using its specificity as a target-specific binding protein. Nickase Cas9 (nCas9) or dead Cas9 (dCas9) modified either one (D10A) or two (D10A/H840A) catalytic residues to lose its cleavage activity. Then, the modified protein was fused with other effector domains such as activators, repressors, or deaminases and used for researches instead of knock-out by DSBs. <br/> Point mutations are the major source of human genetic diseases rather than small insertions/deletions in the genome. To study human genetic disorders animal models are crucial elements. Using typical CRISPR-Cas9 system, inducing single-nucleotide substitutions is challenging because the most DNA DSBs are repaired by error-prone non-homologous end-joining rather than homologous recombination requiring a template donor. Among the recently developed Cas9 variants, base editors, composed of nCas9 or dCas9 and cytidine deaminases, substituting CᆞG to TᆞA or adenine deaminases, substituting AᆞT to GᆞC are powerful tools to induce point mutations effectively without a template donor in target specific manner.<br/> I will describe inducing point mutations using cytidine deaminase in mouse embryos to generate mouse models because the base editing system had not been demonstrated yet in animals. I used Base Editor 3 to induce point mutations in two mouse genes, Dmd and Tyr. I generated a premature stop codon at the target sites efficiently and obtained homozygous mutant F0 mice with high frequencies. <br/> Also, I will describe applying adenine base editors in mouse embryos. Also, I generated a mouse model targeting Tyr gene. However, the base editing systems have a limitation to design proper sgRNAs because of base editing windows where the editing efficiencies were the most effective between 14th and 17th position from protospacer adjacent motif sequence. With extended sgRNAs, I could increase base editing efficiencies in the position out of the canonical base editing windows.<br/>