LAUSR

CRISPR‐Cas9 and Cas12a are widely used sequence‐specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator‐Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI‐dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI‐dCas systems, the PAM (protospacer adjacent motif)‐less SpRY Cas9 variant and the PAM‐relaxed Mb2Cas12a system were explored. Rice cells showed that FokI‐dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI‐dMb2Cas12a system was developed that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single‐chain FokI‐FokI‐dMb2Cas12a system was developed that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR‐Cas systems for genome editing.