PRJNA865310
A new family of CRISPR-type V nucleases with C-rich PAM recognition
Most CRISPR-type V nucleases are stimulated to cleave double-stranded (ds) DNA targets by a35 T-rich PAM which restricts their targeting range. Here, we identify and characterize a new36 family of type V RNA-guided nuclease, Cas12l, that exclusively recognizes a C-rich PAM. The37 organization of genes within its CRISPR locus is similar to type II-B CRISPR-Cas9 systems but38 both sequence analysis and functional studies establish it as a new family of type V effector.39 Biochemical experiments show that Cas12l nucleases function optimally between 37-52C,40 depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases,41 it exhibits collateral non-specific ssDNA and ssRNA cleavage activity that is triggered by42 ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l,43 functions in a heterologous cellular environment, altogether, suggesting that this new group of44 CRISPR associated nucleases may be harnessed as genome editing reagents.