Expanding the targeting scope of FokI-dCas nuclease systems with SpRY and Mb2Cas12a.

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.

Cheng Y ，Sretenovic S ，Zhang Y ，Pan C ，Huang J ，Qi Y ... -  《-》

Applications of Alternative Nucleases in the Age of CRISPR/Cas9.

Guha TK ，Edgell DR 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering.

Saifaldeen M ，Al-Ansari DE ，Ramotar D ，Aouida M ... -  《Cells》

PAM-less plant genome editing using a CRISPR-SpRY toolbox.

Ren Q ，Sretenovic S ，Liu S ，Tang X ，Huang L ，He Y ，Liu L ，Guo Y ，Zhong Z ，Liu G ，Cheng Y ，Zheng X ，Pan C ，Yin D ，Zhang Y ，Li W ，Qi L ，Li C ，Qi Y ，Zhang Y ... -  《-》

Genome Editing in Human Cells Using CRISPR/Cas Nucleases.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. This unit describes protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases.

Wyvekens N ，Tsai SQ ，Joung JK 《-》