Biology subjects

Hibshman, G. N.

Publications and source records attributed to Hibshman, G. N..

3 recordsLinked to original sources

bioRxiv2024

Mechanism of type II-B CRISPR-Cas enhanced specificity

Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature--the REC3 clamp--that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a non-canonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved in type II-B CRISPR-Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the dual mechanisms of high specificity and virulence by FnCas9, with broad implications for biotechnology and therapeutic development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/619592v3_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@8806b0org.highwire.dtl.DTLVardef@6f361eorg.highwire.dtl.DTLVardef@149f506org.highwire.dtl.DTLVardef@757f6d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Unraveling the mechanisms of PAMless DNA interrogation by SpRY Cas9

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an "NGG" protospacer-adjacent motif (PAM) sequence immediately adjacent to the DNA target limits the number of editable genes. To overcome the PAM requirement, a recently developed Cas9 variant, called SpRY-Cas9 was engineered to be "PAMless" (1, 2). However, the molecular mechanisms of how SpRY can recognize all potential PAM sequences and still accurately identify DNA targets have not been investigated. Here, we combined enzyme kinetics, cryo-EM, and single-molecule imaging to determine how SpRY interrogates DNA and recognizes target sites for cleavage. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region of SpRY, which facilitates tight binding to off-target DNA sequences. Once SpRY correctly identifies a target site, nuclease activation occurs [~]1,000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Insights gained from our intermediate structures prompted rationally designed mutants with improved DNA cleavage efficiency. Our findings shed light on the molecular mechanisms of PAMless genome editing with SpRY and provide a framework for the design of future genome editing tools with improved versatility, precision, and efficiency.

biochemistry↗

bioRxiv2022

Modes of inhibition used by phage anti-CRISPRs to evade type I-C Cascade

Cascades are RNA-guided multi-subunit CRISPR-Cas surveillances complexes that target foreign nucleic acids for destruction. Here, we present a 2.9-[A] resolution cryo-electron (cryo-EM) structure of the D. vulgaris type I-C Cascade bound to a double-stranded (ds)DNA target. Our data shows how the 5-TTC-3 protospacer adjacent motif (PAM) sequence is recognized, and provides a unique mechanism through which the displaced, single-stranded non-target strand (NTS) is stabilized via stacking interactions with protein subunits in order to favor R-loop formation and prevent dsDNA re-annealing. Additionally, we provide structural insights into how diverse anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking initial DNA binding. These observations provide a structural basis for directional R-loop formation and reveal how divergent Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.

biochemistry↗