A Cas9 Orthologue-Mediated Cytosine And Adenine Base Editors Recognizing NNAAAA PAN Sequences

Clustered regularly interspaced short palindromic repeat(CRISPR)originated from the bacterial immune system,has been applied as an effective genome targeting technology.By fusing deaminases with Cas9 nickase(n Cas9),various cytosine and adenine base editors(BEs)have been successfully developed,which can induce nucleotide conversions with high efficiency and low indels and install pathogenic single nucleotide variants(SNVs)in cultured cells and animal models.However,the applications of BEs are frequently limited by the specified protospacer adjacent motif(PAM)sequences and protein sizes.To further expand the toolbox for BEs with recognition of novel PAM sequences,we cloned a Cas9 orthologue from Streptococcus sinensis(named as SsiCas9)with smaller size and constructed it into APOBEC1-or APOBEC3A-composed cytosine base editors(CBEs)and Tad A or Tad A*-composed adenine base editors(ABEs),which yield high editing efficiencies,low off-targeting activities,and low indel rates in multiple types of human cells.Relative to PAMless Sp RY Cas9-composed BE4 max,SsiCas9-mediated BE4 max displayed higher editing efficiencies for targets with “NNAAAA” PAM sequences.Moreover,SsiCas9-mediated BE4 max induces highly efficient C-to-T conversions at mouse Ar gene(R841C)to install a human androgen resistance syndrome-related mutation(Ar R820C)in early mouse embryo by microinjection of transcribed RNAs.Thus,we develop novel BEs mediated by a Cas9 orthologue,SsiCas9,and expand toolbox for base conversions and broaden the range of editable genomes in vitro and in vivo.