Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Abstract

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus

“…In summary, we have shown that CONAN, a Cas3-based novel in vitro nucleic acid-detection platform, is a rapid (within 30-40 min), low-cost, and instrument-free detection method for SARS-CoV-2. We also showed that this CONAN-based assay enables single-base-pair discrimination (Figures 2F and 2G) (Yoshimi et al, 2021), thereby facilitating the deployment of CRISPR-dx for quick and mobile POCTs for drug-resistant IAV variants in hospitals or clinics.…”

“…Cascade/Cas3 and Cas12a target sites were based on the human EMX1 gene, the mouse Tyr gene, and the N gene from SARS-CoV-2 (Table S1). A baculovirus expression system was used to purify the EcoCas3 protein, as previously shown (Yoshimi et al, 2021). Briefly, EcoCas3 cDNA was cloned using an octa-histidine tag and a six asparagine-histidine repeat tag into pFastbac-1 plasmids.…”

“…The EcoCascade and crRNA complex were purified in accordance with previously reported methods (Hochstrasser et al, 2014;Jore et al, 2011;Yoshimi et al, 2021). Briefly, EcoCascade/crRNA ribonucleoproteins (RNPs) were expressed in JM109 (DE3) by co-transformation with three plasmids: one plasmid encoding a hexahistidine tag and HRV3C protease recognition site in the N-terminus of Cas11 (plasmid pCDFDuet-1); one plasmid containing the EcoCascade operon and the genes encoding Cas5, Cas6, Cas7, Cas8, and Cas11 (plasmid pRSFDuet-1); and the final plasmid encoding crRNA (pACYCDuet-1) (Yoshet al, 2021).…”

“…Interestingly, Streptococcus thermophiles Cas3 (Sinkunas et al, 2011) and Methanocaldococcus jannaschii Cas3 enzymes (Beloglazova et al, 2011) have an indiscriminate type of single-stranded DNA (ssDNA) cleavage when activated by Mg 2+ bound to the catalytic site of the HD domain. In contrast, activation of the HD domain in Escherichia coli Cas3 (EcoCas3) (Mulepati and Bailey, 2013;Yoshimi et al, 2021) and Thermus thermophilus Cas3 (Mulepati and Bailey, 2011) is elicited by transition metal ions such as Co 2+ and Ni 2+ , but not by Ca 2+ and Mg 2+ . Despite type I-E E. coli CRISPR being one of the most thoroughly biochemically characterized in vitro plasmid DNA degradation-inducing systems (Hidalgo-Cantabrana and Barrangou, 2020;Zheng et al, 2020), whether the CRISPR-Cas3 system can mediate the target-activated, nonspecific ssDNA cleavage reported for Cas12 and Cas13 (Chen et al, 2018;Gootenberg et al, 2017) remains an open question.…”

“…Although we have discovered the collateral ssDNA cleavage activity in the EcoCascade-Cas3 system, this study does not describe the mechanisms underlying how Cas3 mediates the collateral ssDNA cleavages as well as targeted double-stranded DNA cleavages. Our preprint manuscript describes several insights for these molecular mechanisms (Yoshimi et al, 2021).…”