7lyt

Publication Abstract from PubMed

CRISPR-CasPhi, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasPhi (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasPhi that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasPhi binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.

DNA interference states of the hypercompact CRISPR-CasPhi effector.,Pausch P, Soczek KM, Herbst DA, Tsuchida CA, Al-Shayeb B, Banfield JF, Nogales E, Doudna JA Nat Struct Mol Biol. 2021 Aug;28(8):652-661. doi: 10.1038/s41594-021-00632-3. , Epub 2021 Aug 11. PMID:34381246^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.