PAM recognition by miniature CRISPR-Cas14 triggers programmable double-stranded DNA cleavage

TLDR: It is demonstrated that a T-rich PAM sequence triggers Cas14 proteins to also cut double-stranded DNA generating staggered ends, paving the way for genome editing applications with Cas14s.

Abstract: Small and robust CRISPR-Cas nucleases are highly desirable for genome editing applications. Being guided by a RNA to cleave targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, smaller more compact versions would simplify delivery and extend application. Recently, a new class 2 system encoding a miniature (529 amino acids) effector, Cas14a1, has been shown to exclusively function as a PAM-independent single stranded DNA nuclease. Using biochemical methods, we show that a T-rich PAM sequence triggers Cas14 proteins to also cut double-stranded DNA generating staggered ends. Finally, we demonstrate the ability of Cas14a1 to target and cleave cellular human chromosomal DNA paving the way for genome editing applications with Cas14s.