Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble

Abstract

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T . thermophilus ) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis ( S. epidermidis ) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.

Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation

“…3A). Like in Type I complexes, the crRNA is presented by the effector complex in discontinuous segments for base-pairing with the target nucleic acid (11)(12)(13)74). In addition, each Csm3/Cmr4 inserts a "finger" loop into the duplex, flipping out every 6 th base pair (11)(12)(13)(14).…”

“…Unlike in Type I effectors, the Cas7-like subunits of Type III effector complexes (Csm3/Cmr4) are catalytically active. Cleavage requires a conserved aspartate (Asp) residue in Csm3/Cmr4 and occurs 3´ to every flipped base, resulting in a characteristic six-nucleotide cleavage periodicity (11)(12)(13)(14)74). RNA cleavage requires a 2´-OH in the target RNA for cleavage, and the resulting termini of the reaction products have a 5´-OH and either a 3´-phosphate or 2´,3´cyclic phosphate (14,81).…”

“…DNA cleavage is catalyzed by Cas10's HD nuclease domain and requires RNA binding, but not RNA cleavage by the effector complex (26,77,(87)(88)(89). The HD domain cleaves random sequences of ssDNA and generally requires transition metals (Ni 2+ or Mn 2+) for maximal activity, similar to Cas3 (26,74,77,(86)(87)(88). How Cas10 is activated to bind and cleave ssDNA is not clear, as ssDNA could not be visualized in structures of Csm/Cmr and few conformational changes were observed in the HD domain upon RNA binding (12,13,74).…”

“…The HD domain cleaves random sequences of ssDNA and generally requires transition metals (Ni 2+ or Mn 2+) for maximal activity, similar to Cas3 (26,74,77,(86)(87)(88). How Cas10 is activated to bind and cleave ssDNA is not clear, as ssDNA could not be visualized in structures of Csm/Cmr and few conformational changes were observed in the HD domain upon RNA binding (12,13,74). The identification of Cas10 mutations that constitutively activate ssDNA cleavage indicate that RNA binding may relieve an autoinhibited state (13).…”

Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific Viral Diagnostic

“…pCDF-5xT7-TtCsm was used as a template for site-directed mutagenesis to mutate the Csm3 residue D33 to alanine (D33A) to inactivate Csm3-mediated cleavage of target RNA (pCDF-5xT7-TtCsm Csm3-D34A ) (Liu et al, 2017). The CRISPR array in pACYC-TtCas6-4xcrRNA4.5 (Addgene plasmid # 127764) (Liu et al, 2019) was replaced with a synthetic CRISPR array (GeneArt) containing five repeats and four identical spacers, designed to target the N-gene of SARS-CoV2 (i.e., pACYC-TtCas6-4xgCoV2N1). The nuclease TtCsm6 was expressed from pC0075 TtCsm6 His6-TwinStrep-SUMO-BsaI (Addgene plasmid # 115270) (Gootenberg et al, 2018).…”