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Title: Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a

Abstract

CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.

Authors:
ORCiD logo [1];  [1];  [1];  [1];  [2];  [1]; ORCiD logo [3];  [4];  [1];  [1];  [1]; ORCiD logo [5]; ORCiD logo [6]; ORCiD logo [7]
  1. Univ. of California, Berkeley, CA (United States)
  2. Gladstone Inst., San Francisco, CA (United States)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  5. Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  6. Gladstone Inst., San Francisco, CA (United States); Univ. of California, San Francisco, CA (United States)
  7. Univ. of California, Berkeley, CA (United States); Gladstone Inst., San Francisco, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1560609
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
eLife
Additional Journal Information:
Journal Volume: 8; Journal ID: ISSN 2050-084X
Publisher:
eLife Sciences Publications, Ltd.
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Knott, Gavin J., Cress, Brady F., Liu, Jun-Jie, Thornton, Brittney W., Lew, Rachel J., Al-Shayeb, Basem, Rosenberg, Daniel J., Hammel, Michal, Adler, Benjamin A., Lobba, Marco J., Xu, Michael, Arkin, Adam P., Fellmann, Christof, and Doudna, Jennifer A. Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a. United States: N. p., 2019. Web. doi:10.7554/eLife.49110.
Knott, Gavin J., Cress, Brady F., Liu, Jun-Jie, Thornton, Brittney W., Lew, Rachel J., Al-Shayeb, Basem, Rosenberg, Daniel J., Hammel, Michal, Adler, Benjamin A., Lobba, Marco J., Xu, Michael, Arkin, Adam P., Fellmann, Christof, & Doudna, Jennifer A. Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a. United States. doi:10.7554/eLife.49110.
Knott, Gavin J., Cress, Brady F., Liu, Jun-Jie, Thornton, Brittney W., Lew, Rachel J., Al-Shayeb, Basem, Rosenberg, Daniel J., Hammel, Michal, Adler, Benjamin A., Lobba, Marco J., Xu, Michael, Arkin, Adam P., Fellmann, Christof, and Doudna, Jennifer A. Fri . "Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a". United States. doi:10.7554/eLife.49110. https://www.osti.gov/servlets/purl/1560609.
@article{osti_1560609,
title = {Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a},
author = {Knott, Gavin J. and Cress, Brady F. and Liu, Jun-Jie and Thornton, Brittney W. and Lew, Rachel J. and Al-Shayeb, Basem and Rosenberg, Daniel J. and Hammel, Michal and Adler, Benjamin A. and Lobba, Marco J. and Xu, Michael and Arkin, Adam P. and Fellmann, Christof and Doudna, Jennifer A.},
abstractNote = {CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.},
doi = {10.7554/eLife.49110},
journal = {eLife},
number = ,
volume = 8,
place = {United States},
year = {2019},
month = {8}
}

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Cited by: 2 works
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Figures / Tables:

Figure 1 Figure 1: Overall structure of the LbCas12a-crRNA-AcrVA4 complex. (A) Schematic representation of Cas12a activity. (B) Unrooted maximum likelihood phylogenetic tree of Type V-A CRISPR-Cas12a. Species known to be susceptible or unsusceptible to phage-derived AcrVA4 are highlighted. The triangle denotes collapsed branches of Cas12b-e. (C) Schematic representation of LbCas12a and themore » mature crRNA modeled within the cryo-EM structures. (D) Two views of the LbCas12a-crRNA complex (cartoon) bound to AcrVA4 (surface) shown related by a 180˚ rotation. The color scheme for the crRNA, LbCas12a, and AcrVA4 in panels B, C, and D are used throughout the manuscript.« less

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