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.
A previously unknown mechanism that steers Cas9 catalysis is uncovered and the potential to improve Cas9 fidelity by modulating guide repeat interactions is demonstrated.
Ramadevi Chilamkurthy, Sruthi Sudhakar, Adrian A. Pater et al.· Nucleic Acids Research· 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
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
A PLM-assisted physics-driven approach is presented that utilizes atomistic molecular dynamics simulations and automated path searching to efficiently obtain the complete kinetic insights, including the transition state structures, for the conformational changes of Cas before DNA cleavage.
This review uniquely addresses that gap by presenting an integrated framework that connects molecular design principles with clinical applicability in gRNAs 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
A protein design strategy is used that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs, establishing a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.
Petr Skopintsev, Isabel Esain-Garcia, Evan C. DeTurk et al.· Science· 2 citations
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