LAUSR

CRISPR/Cas9 system has been applied as an effective genome‐targeting technology. By fusing deaminases with Cas9 nickase (nCas9), various cytosine and adenine base editors (CBEs and ABEs) have been successfully developed that can efficiently induce nucleotide conversions and install pathogenic single nucleotide variants (SNVs) in cultured cells and animal models. However, the applications of BEs are frequently limited by the specific protospacer adjacent motif (PAM) sequences and protein sizes. To expand the toolbox for BEs that can recognize novel PAM sequences, we cloned a Cas9 ortholog from Streptococcus sinensis (named as SsiCas9) with a smaller size and constructed it into APOBEC1‐ or APOBEC3A‐composed CBEs and TadA or TadA*‐composed ABEs, which yield high editing efficiencies, low off‐targeting activities, and low indel rates in human cells. Compared to PAMless SpRY Cas9‐composed BE4max, SsiCas9‐mediated BE4max displayed higher editing efficiencies for targets with “NNAAAA” PAM sequences. Moreover, SsiCas9‐mediated BE4max induced highly efficient C‐to‐T conversions in the mouse Ar gene (R841C) to introduce a human androgen resistance syndrome‐related mutation (AR R820C) in early mouse embryos. Thus, we developed novel BEs mediated by SsiCas9, expanded the toolbox for base conversions, and broadened the range of editable genomes in vitro and in vivo.