Discovery and engineering of Tsp2Cas9 for genome editing

The CRISPR-Cas9 system stands as a potent genome-editing tool deployed across various organisms 1 . Within this system, a guide RNA (gRNA) collaborates with a Cas9 nuclease, forming a complex wherein the gRNA specifies the target site, and the Cas9 executes DNA cleavage 2 . Additionally, base editors have been developed by coupling a nickase form of Cas9 nuclease (Cas9n) with a deaminase, enabling base alterations at the target sites 3 . Recently, Cas9n was fused with an engineered reverse transcriptase (RT) enzyme to enable the development of prime editors capable of generating genomic changes over large stretches of base pairs 4 . However, a notable constraint of CRISPR-Cas9 system lies in its reliance on protospacer adjacent motif (PAM) sequences at target sites, limiting its scope of targeting 2 . Public databases harbour thousands of computationally identified Cas9 orthologs, each characterized by unique PAM requirements 5 . By developing new Cas9 genome-editing tools, it is becoming feasible to edit a broader array of genomic sites.