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CRISPR-Cas Endonucleses

Efficient and site-specific genome engineering can be achieved based on programmable dsDNA cleavage using CRISPR-Cas systems. Structural studies on single-component Cas and multi-component Cascade complexes are shedding light on the principles underlying cleavage chemistry of dsDNA and RNA targets. Future challenges include an understanding of the diverse mechanisms adopted by distinct CRISPR-Cas systems in efforts to broaden and enhance their applicability as genome editing tools. Efforts are also underway to provide a structural understanding of recognition principles involving evolved bacteriophage suppressor proteins that inhibit the CRISPR-Cas pathway, thereby regulating the genome engineering activities of CRISPR-Cas systems.


Cpf1 and C2c1 Endoribonucleses

The earlier research on the dsDNA cleavage activity of class II type II Cas9 endoribonucleases has been recently extended to class II type V Cpf1 and C2c1 counterparts. The Cpf1 and C2c1 are distinctly different from Cas9 since (1) Cpf1 and C2c1 contain only one RuvC domain but lack the HNH domain observed in Cas9; (2) Cpf1 is guided by a single crRNA, while C2c1 and Cas9 also require tracrRNA; (3) Cpf1 and C2c1 recognize distal 5’-T-rich PAMs, in contrast to a proximal 3′-G-rich PAM recognition by Cas9; and (4) Cpf1 and C2c1 make staggered double-strand breaks, whereas Cas9 generates blunt ends. We have undertaken structural studies on ternary complexes of Cpf1 and C2c1 with guide RNA and target dsDNA in efforts to provide a molecular basis for efforts towards harnessing these endoribonucleases for genome editing in mammalian cells.


PAM-dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease

PAM-dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease

C2c1 is a newly-identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering-up of 20-bp RNA-DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a “locked” conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas9 systems, thereby broadening and enhancing their applicability as genome editing tools.

Yang, H., Gao, P., Rajashankar, K. R., & Patel, D. J. (2017). PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease. Cell 167,1814-1828.

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

CRISPR-Cas9 and CRISPR-Cpf1 systems have been successfully harnessed for genome editing. In the CRISPR-Cas9 system, the preordered A-form RNA seed sequence and preformed protein PAM-interacting cleft are essential for Cas9 to form a DNA recognition-competent structure. Whether the CRISPR-Cpf1 system employs a similar mechanism for target DNA recognition remains unclear. Here, we have determined the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with crRNA and target DNA, in agreement with a recently reported structure of the same complex. Structural comparison between the AsCpf1-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpf1 (LbCpf1)-crRNA binary complex identifies a unique mechanism employed by Cpf1 for target recognition. The seed sequence required for initial DNA interrogation is disordered in the Cpf1-cRNA binary complex, but becomes ordered on ternary complex formation. Further, the PAM interacting cleft of Cpf1 undergoes an “open-to-closed” conformational change upon target DNA binding, which in turn induces structural changes within Cpf1 to accommodate the ordered A-form seed RNA segment. This unique mechanism of target recognition by Cpf1 is distinct from that reported previously for Cas9.

Gao, P., Yang, H., Rajanshankar, K. R., Huang, Z. & Patel, D. J. (2016). Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target recognition. Cell Research 26, 901-913.