Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, United States; Graduate Group in Biophysics, University of California, Berkeley, Berkeley, United States
Michal Hammel
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, United States
Benjamin A Adler
UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, United States; Department of Bioengineering, University of California, Berkeley, Berkeley, United States
Marco J Lobba
Department of Chemistry, University of California, Berkeley, Berkeley, United States
Michael Xu
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
Department of Bioengineering, University of California, Berkeley, Berkeley, United States; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, United States
Gladstone Institutes, San Francisco, United States; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; Gladstone Institutes, San Francisco, United States; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, United States; Department of Chemistry, University of California, Berkeley, Berkeley, United States; Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, United States
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.
