CRISPR RNA-guided DNA cleavage by reconstituted Type I-A immune effector complexes

Abstract

Diverse CRISPR-Cas immune systems protect archaea and bacteria from viruses and other mobile genetic elements. All CRISPR-Cas systems ultimately function by sequence-specific destruction of invading complementary nucleic acids. However, each CRISPR system uses compositionally distinct crRNP [CRISPR (cr) RNA/Cas protein] immune effector complexes to recognize and destroy invasive nucleic acids by unique molecular mechanisms. Previously, we found that Type I-A (Csa) effector crRNPs from Pyrococcus furiosus function in vivo to eliminate invader DNA. Here, we reconstituted functional Type I-A effector crRNPs in vitro with recombinant Csa proteins and synthetic crRNA and characterized properties of crRNP assembly, target DNA recognition and cleavage. Six proteins (Csa 4–1, Cas3″, Cas3′, Cas5a, Csa2, Csa5) are essential for selective target DNA binding and cleavage. Native gel shift analysis and UV-induced RNA–protein crosslinking demonstrate that Cas5a and Csa2 directly interact with crRNA 5′ tag and guide sequences, respectively. Mutational analysis revealed that Cas3″ is the effector nuclease of the complex. Together, our results indicate that DNA cleavage by Type I-A crRNPs requires crRNA-guided and protospacer adjacent motif-dependent target DNA binding to unwind double-stranded DNA and expose single strands for progressive ATP-dependent 3′–5′ cleavage catalyzed by integral Cas3′ helicase and Cas3″ nuclease crRNP components.

Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

“…Recently, it was also demonstrated that like some other CARF-domain nucleases (see above), also Csx1 of P. furiosus was endowed with ring nuclease activity, self-inactivating its cOA activators [147]. Apart from additional biochemical studies focusing on the two other type I effector complexes in P. furiosus [80,81], in vivo immunity against engineered plasmid invaders was shown for all effector complexes independently by analyzing a plethora of cas (and accessory genes) mutants [122,147,179]. Recently P. furious has also become a model to study spacer acquisition, revealing that new extrachromosomal spacers are preferentially acquired from broken DNA ends, supplied by plasmids with rolling-circle rather than theta replication [33].…”

“…CRISPR type I systems in general employ a multi-subunit effector complex termed CRISPR-associated complex for antiviral defense (Cascade) which, upon PAM recognition and crRNA hybridization catalyzes protospacer degradation by the action of the Cas3 enzyme endowed with ssDNA-endonuclease (provided by an HD domain) and helicase activity. Cas3 nicks the strand opposite to the crRNA-protospacer heteroduplex and is then activated to processively cleave the upstream region [73][74][75][76][77][78][79][80][81] (c.f. Figure 1).…”

RNA: interactions drive functionalities

“…CrRNAs constitute prokaryotic immune system by assisting Cas proteins recognize and cut exogenous DNA. On phage infection, proteo-spacers are lysed by bacteria Cas protein complexes and integrated into the 5′ end of the CRISPR sites in host genome which are then transcribed into crRNAs and function as the template against phage proto-spacers once infected again [62][63][64].…”

CRISPR/Cas System for Genome Editing: Progress and Prospects as a Therapeutic Tool

“…Cas3 and Cas10 are considered as the signature genes for types I and III, respectively (Shmakov et al, 2017). Type III (B) Cmr is probably rare since it has been found to cleave targeted RNAs (Majumdar and Terns, 2019).…”