TY - JOUR
T1 - DNA interference states of the hypercompact CRISPR–CasΦ effector
AU - Pausch, Patrick
AU - Soczek, Katarzyna M.
AU - Herbst, Dominik A.
AU - Tsuchida, Connor A.
AU - Al-Shayeb, Basem
AU - Banfield, Jillian F.
AU - Nogales, Eva
AU - Doudna, Jennifer A.
N1 - Funding Information:
We thank E. Charles, C. Huang, G. Knott, D. Smock and members of the Doudna and Nogales laboratories for discussion. We thank J. Cofsky and G. Knott for critical reading and comments on the manuscript. We thank J. Remis, D. Toso and G. Knott for electron microscopy assistance and A. Chintangal for computational support. EM data were collected at the Cal-Cryo facility located at UC Berkeley. P.P. is supported by the NIH Somatic Cell Genome Editing consortium (NIH U01AI142817-02). C.A.T. is supported by Campus Executive Grants 2101705 and 1655264 through Sandia National Laboratories. B.A.-S. was supported by an NSF Graduate Research Fellowship (DGE 1752814). J.A.D. receives funding from the Centers for Excellence in Genomic Science of the National Institutes of Health under award number RM1HG009490, from the Somatic Cell Genome Editing Program of the Common Fund of the National Institutes of Health under award number U01AI142817-02, and from the National Science Foundation under award number 1817593. D.A.H. was supported by the EMBO (ALTF 1002-2018) and SNSF (P2BSP3_181878). J.A.D. and E.N. are Howard Hughes Medical Institute Investigators.
Publisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.
PY - 2021/8
Y1 - 2021/8
N2 - CRISPR–CasΦ, 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 CasΦ (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 CasΦ 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 CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.
AB - CRISPR–CasΦ, 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 CasΦ (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 CasΦ 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 CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.
UR - http://www.scopus.com/inward/record.url?scp=85112300011&partnerID=8YFLogxK
U2 - 10.1038/s41594-021-00632-3
DO - 10.1038/s41594-021-00632-3
M3 - Article
C2 - 34381246
AN - SCOPUS:85112300011
SN - 1545-9993
VL - 28
SP - 652
EP - 661
JO - Nature Structural & Molecular Biology
JF - Nature Structural & Molecular Biology
IS - 8
ER -