TY - JOUR

T1 - Biochemical plasticity of the Escherichia coli CRISPR Cascade revealed by in vitro reconstitution of Cascade activities from purified Cas proteins

AU - Lemak, Sofia

AU - Brown, Greg

AU - Makarova, Kira S.

AU - Koonin, Eugene V.

AU - Yakunin, Alexander

N1 - © 2024 Federation of European Biochemical Societies.

PY - 2024

Y1 - 2024

N2 - The most abundant clustered regularly interspaced short palindromic repeats (CRISPR) type I systems employ a multisubunit RNA-protein effector complex (Cascade), with varying protein composition and activity. The Escherichia coli Cascade complex consists of 11 protein subunits and functions as an effector through CRISPR RNA (crRNA) binding, protospacer adjacent motif (PAM)-specific double-stranded DNA targeting, R-loop formation, and Cas3 helicase-nuclease recruitment for target DNA cleavage. Here, we present a biochemical reconstruction of the E. coli Cascade from purified Cas proteins and analyze its activities including crRNA binding, dsDNA targeting, R-loop formation, and Cas3 recruitment. Affinity purification of 6His-tagged Cas7 coexpressed with untagged Cas5 revealed the physical association of these proteins, thus producing the Cas5-Cas7 subcomplex that was able to bind specifically to type I-E crRNA with an efficiency comparable to that of the complete Cascade. The crRNA-loaded Cas5-7 was found to bind specifically to the target dsDNA in a PAM-independent manner, albeit with a lower affinity than the complete Cascade, with both spacer sequence complementarity and repeat handles contributing to the DNA targeting specificity. The crRNA-loaded Cas5-7 targeted the complementary dsDNA with detectable formation of R-loops, which was stimulated by the addition of Cas8 and/or Cas11 acting synergistically. Cascade activity reconstitution using purified Cas5-7 and other Cas proteins showed that Cas8 was essential for specific PAM recognition, whereas the addition of Cas11 was required for Cas3 recruitment and target DNA nicking. Thus, although the core Cas5-7 subcomplex is sufficient for specific crRNA binding and basal DNA targeting, both Cas8 and Cas11 make unique contributions to efficient target recognition and cleavage.

AB - The most abundant clustered regularly interspaced short palindromic repeats (CRISPR) type I systems employ a multisubunit RNA-protein effector complex (Cascade), with varying protein composition and activity. The Escherichia coli Cascade complex consists of 11 protein subunits and functions as an effector through CRISPR RNA (crRNA) binding, protospacer adjacent motif (PAM)-specific double-stranded DNA targeting, R-loop formation, and Cas3 helicase-nuclease recruitment for target DNA cleavage. Here, we present a biochemical reconstruction of the E. coli Cascade from purified Cas proteins and analyze its activities including crRNA binding, dsDNA targeting, R-loop formation, and Cas3 recruitment. Affinity purification of 6His-tagged Cas7 coexpressed with untagged Cas5 revealed the physical association of these proteins, thus producing the Cas5-Cas7 subcomplex that was able to bind specifically to type I-E crRNA with an efficiency comparable to that of the complete Cascade. The crRNA-loaded Cas5-7 was found to bind specifically to the target dsDNA in a PAM-independent manner, albeit with a lower affinity than the complete Cascade, with both spacer sequence complementarity and repeat handles contributing to the DNA targeting specificity. The crRNA-loaded Cas5-7 targeted the complementary dsDNA with detectable formation of R-loops, which was stimulated by the addition of Cas8 and/or Cas11 acting synergistically. Cascade activity reconstitution using purified Cas5-7 and other Cas proteins showed that Cas8 was essential for specific PAM recognition, whereas the addition of Cas11 was required for Cas3 recruitment and target DNA nicking. Thus, although the core Cas5-7 subcomplex is sufficient for specific crRNA binding and basal DNA targeting, both Cas8 and Cas11 make unique contributions to efficient target recognition and cleavage.

KW - Cas proteins; Cascade;CRISPR; Escherichia coli;RNA/DNA binding

U2 - 10.1111/febs.17295

DO - 10.1111/febs.17295

M3 - Article

C2 - 39375921

SP - 1

EP - 18

JO - Febs Journal

JF - Febs Journal

SN - 1742-464X

ER -