Oral Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Three distinct modes of CRISPR-Cas12a inactivation (#43)

Gavin J. Knott 1 , Brittney W. Thornton 1 , Marco J. Lobba 2 , Jun-Jie Liu 1 , Kyle E. Watters 1 , Jennifer A. Doudna 1 2 3 4 5 6
  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
  2. Department of Chemistry, University of California, Berkeley, CA, USA
  3. Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
  4. Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA
  5. Howard Hughes Medical Institute, University of California, Berkeley, CA, USA
  6. Innovative Genomics Institute, University of California, Berkeley, CA, USA

Bacteria and archaea protect themselves against bacteriophage using CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) adaptive immunity1. In their native context, Cas enzymes can be inhibited by bacteriophage-derived anti-CRISPR proteins (Acrs) to thwart CRISPR adaptive immune systems, comprising the hosts defences and enabling phage replication2. Recently, bacteriophage-derived Acrs were discovered that inhibit the Class 2 type V-A effector nuclease Cas12a3.

Cas12a is a subtype of Cas-effector harnessed as a robust tool for genome editing, laboratory diagnostics, and nucleic acid detection4. Unlike Cas9 which targets double-stranded DNA (dsDNA) and cleaves with single-turnover kinetics, Cas12a is guided by an RNA to target either single-stranded or dsDNA activating a multiple turnover DNase5. We show that the newly discovered inhibitors (AcrVA1, AcrVA4, and AcrVA5) disable Cas12a using three functionally distinct mechanisms, including two previously unobserved strategies. AcrVA4 and AcrVA5 block target dsDNA recognition, where AcrVA4 drives Cas12a dimerization and forces DNA release prior to target cleavage. In contrast, AcrVA1 is a multiple-turnover broad-spectrum inhibitor that triggers endoribonucleolytic cleavage of the RNA guide recognition sequence to irreversibly inactivate Cas12a. These distinct mechanisms equip bacteriophage with potent tools to evade CRISPR-Cas DNA targeting and support biotechnological applications where enzymatic inhibition of Cas12a or rapid removal of DNA-bound Cas12a are desirable. 

 

 

  1. Wright A.V. et al., (2016) Biology and applications of CRISPR systems: Harnessing natures toolbox for genome engineering. Cell. 164, 29-43.
  2. Borges A.L. et al., (2018) Bacteriophage cooperation suppresses CRISPR-Cas3 and Cas9 immunity. Cell. 174, 917-925.
  3. Watters K.E. et al., (2018) Systematic discovery of natural CRISPR-Cas12a inhibitors. Science. 10.1126/science.aau5138.
  4. Knott G.J. and Doudna J.A., (2018) CRISPR-Cas guides the future of genetic engineering. Science. 361, 866-869.
  5. Chen J.S. et al., (2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 360, 436-439.