LAUSR

Dear Editor, Most genetic diseases in humans are caused by singlenucleotide mutations. Although genome editing with either the CRISPR-based cytosine base editor (CBE) or the adenine base editor (ABE) holds great promise for gene correction of C-to-T and A-to-G base substitutions in some genetic diseases, both editors are useless for correction of other variants such as base transversion, small insertions and deletions (indels). The prime editing system, a “search-and replace” genome editing technology, was recently added to the genome editing toolkit. The prime editors (PEs) combine an exogenous CRISPR/Cas9 system and endogenous DNA repair system to achieve an increased range of editing versatility, induces all types of base-to-base conversions out of CBE and ABE (C→T, G→A, A→G, and T→C), small indel, and their combinations. The prime editing system evolved from PE1 to PE3 (PE3b) with stepwise efficiency improvement. The executor of PE1 was constructed by fusing an engineered Cas9 nickase with a reverse transcriptase (M-MLV RTase), which can target genome sites, nick DNA, and trigger reverse transcription (RT). The executor combining with the engineered prime editing guide RNA (pegRNA) searches for and nicks the target DNA, and thus, new genetic information is encoded into genome by RT. Then, mutations were introduced to M-MLV RTase to improve the editing efficiency of PE1, which is referred to as PE2. Subsequently, in the PE3 system, to further improve editing efficiency, an additional sgRNA is used to induce nick on the non-edited strand to trigger the endogenous mismatch repair pathway. In comparison with base editors, PE induces base institutions in more extended regions with fewer bystander mutations. With its unique versatility and accuracy, this technology broadens the scope of genome editing and opens a new avenue for targeted mutagenesis and gene correction in many organisms. However, the efficiency of PE was reported only in five different cell types; it has not been investigated in animals. Here, we demonstrate that PE can be employed to generate mutant mice with singlenucleotide substitutions. We first validated the editing versatility of PEs in human HEK293T cells at eight loci (Supplementary Table S1), including two loci (RUNX1 and RNF2) that were reported by Anzalone et al. PE3 was selected for gene targeting validation, due to its higher editing efficiency compared with PE2. Sanger sequencing revealed that PE3 induced significant base conversions at six (RUNX1, RNF2, EMX1, VEGFA, SRD5A3, and KCNA1) out of eight targeted sites (Supplementary Fig. S1a, b). PE3 was then used to induce point mutations in the X-linked androgen receptor (Ar) gene and the homeobox protein Hox-D13 (Hoxd13) gene in mouse neuro-2a (N2a) cells. Both targeted mutations in mice are homologous to human variants associated with clinical diseases in ClinVar. pegRNAs and nick-editing sgRNAs targeting these two genes were designed (Supplementary Table S2). We designed pegRNAs starting with a primer binding site (PBS) length of 13 nt and an RT template length of ~13–15 nt. Nicks were positioned 3′ of the edit ~40–60 bp from the pegRNA-induced nick. Sanger sequencing revealed that PE3 efficiently (~8–40%) mediated base transversions at three target sites of Hoxd13 and Ar (pegHoxd13-1 for G to C, pegHoxd13-2 for G to T, pegAr-2 for G to T) (Fig. 1a;