A previously unknown mechanism that steers Cas9 catalysis is uncovered and the potential to improve Cas9 fidelity by modulating guide repeat interactions is demonstrated.
Abstract
Abstract A widely adopted modification of CRISPR–Cas9 is fusion of the naturally occurring two-component dual guide RNA (dgRNA) to create an artificial single guide RNA (sgRNA). Here we find that these guide architectures induce differential catalysis, gene editing, and specificity. Spacer sequence and RNA structural features could not predict guide architecture editing preference across 255 endogenous targets. We used cryo-EM and molecular dynamics to identify a new Cas9 structural motif, the guide repeat clasp (GRC), that checks guide RNA repeat structure and coordinates with R-loop sensing checkpoint mechanisms to help license cleavage. Limited mutagenesis of GRC residues significantly altered Cas9 editing and specificity, supporting a key role in catalysis. To further understand the role of the GRC and guide RNA repeat dynamics, we created guide repeat-truncated sgRNAs, or grtRNAs, which conferred some dgRNA properties onto sgRNA, including generally lower off-target editing for targets with PAM-proximal mismatches. dgRNAs and grtRNAs could be combined with a new high-fidelity Cas9 variant called ZiFY, rationally designed to reduce editing of targets with PAM-distal mismatches, to generate broader mismatched target discrimination. These results uncover a previously unknown mechanism that steers Cas9 catalysis and demonstrate the potential to improve Cas9 fidelity by modulating guide repeat interactions.
Cas12a is highly accommodative toward noncanonical activation pathways to the extent of flipping its identity to be a DNA-guided RNA-targeting effector. A sequence engineering approach was used to systematically identify desirable guide DNA (gDNA) sequence motifs to achieve comparable RNA targeting efficiency as the canonical RNA-guided Cas12a with good selectivity down to single-nucleotide mismatch. Importantly, we introduced a split gDNA design concept with greater energetic differences arising from subtle nucleotide changes to probe the key spacer features for effective Cas12a-gDNA activation. Similar to the canonical RNA-guided activation pathway, Cas12a was found to engage actively in the "seed-like" scaffold-proximal region while the scaffold-distal region was largely hybridization-driven. We further evolved the split gDNA design to enhance the sequence selectivity by up to 21-fold compared to a single gDNA design and achieve single-nucleotide discrimination among representative let-7 family members. This study has established a gDNA sequence design framework to reprogram Cas12a as a precise RNA targeting platform.
CRISPR–Cas9 has revolutionised genome editing by enabling efficient and programmable modification of defined DNA sequences, with guide RNAs (gRNAs) serving as indispensable elements that direct Cas9 to specific genomic loci. Initially regarded as auxiliary components, gRNAs are now recognized as critical determinants of editing efficiency and specificity and have attracted growing attention as independent targets for engineering. Chemical modification, sequence optimisation, and structural alteration of gRNAs have been shown to enhance on‐target activity, suppress off‐target effects and cytotoxicity, and even achieve allele‐selective precision editing in a programmable manner. Moreover, advances in artificial intelligence and machine learning have markedly improved the predictive accuracy of gRNA design through large‐scale data analysis. Despite rapid progress, a consolidated review that integrates chemical, structural, and computational advances in gRNA engineering and highlights their translational potential for therapeutic genome editing has been lacking. This review uniquely addresses that gap by presenting an integrated framework that connects molecular design principles with clinical applicability.
Masaki Kawamata, S. Niwa, Atsushi Suzuki· Chemical Biology and Drug De...· 0 citations
RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection and applies it to detect single-point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems.
I. Iwe, Frank X. Liu, A. Corsano et al.· Nucleic Acids Research· 0 citations
It is shown that short RNAs can directly occupy the canonical crRNA-binding channel and trigger a catalytically competent trans cleavage state in the absence of PAM recognition or canonical R-loop formation.
I. Iwe, S. Singh, K. Guan et al.· medRxiv· 0 citations
The mechanisms of nuclease activation are explored by solving seven ternary cryo-electron mi-croscopy structures of wild-type Cas13d in complex with matched and mismatched targets and an active site loop in the HEPN domains that regulates substrate accessibility is identified.
Chia-Wei Chou, Selma Sinan, Hung-Che Kuo et al.· bioRxiv· 0 citations
It is demonstrated that local nucleosome sequence and structure profoundly influence Cas nuclease accessibility and specificity, with HIFIv1 emerging as the top-performing nuclease for nucleosomal targets, while evoSpCas9 excelled in exposed contexts.
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