4.7 Review

Constructing next-generation CRISPR-Cas tools from structural blueprints

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Summary: This study reports a new CRISPR-Cas3 system that can create targeted large deletions in the human genome. RNP delivery of Cas3 nuclease and target recognition complex can achieve editing efficiency of up to 95%. Furthermore, the study demonstrates that supplying cas11 is a universal strategy to enable different types of CRISPR-Cas3 editors and expands our ability to engineer long-range genome edits.

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Summary: In this study, the structure of Cas9 during mismatch cleavage was determined using kinetics-guided cryo-electron microscopy. It was found that a linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, preventing Cas9 activation. Additionally, mismatches distal to the protospacer adjacent motif were stabilized by reorganization of a loop in the RuvC domain. Mutations of mismatch-stabilizing residues reduced off-target DNA cleavage while maintaining rapid on-target DNA cleavage. This study provides proof of concept for the design of next-generation high-fidelity Cas9 variants targeting mismatch tolerance regions.

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Summary: Researchers have discovered that the Cas9 protein bends and twists DNA when binding to PAM, allowing DNA nucleotides to be extracted from the double helix and examined by the guide RNA. This finding is significant for understanding the efficiency of genome editing.

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Summary: This article reviews the role of molecular simulations in the CRISPR-Cas9 revolution, including studies on RNA binding, catalytic mechanism, off-target effects, etc. It reveals the dynamics and mechanism of CRISPR-Cas9, contributing to understanding its function and specificity.

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Summary: The crystallographic structures of Cas9 bound to off-target substrates reveal that noncanonical base-pairing interactions within the guide:off-target heteroduplex enable off-target binding. Single-nucleotide deletions in off-target substrates are accommodated by base skipping or multiple noncanonical base pairs. PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe. These insights contribute to the improved rational design of guide RNAs and off-target prediction algorithms.
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Summary: Since the discovery of CRISPR-Cas9, labs worldwide have used this gene editing tool to modify the genomes of living cells. Scientists and engineers have altered Cas9 to make gene editing more precise and efficient. Key aspects of Cas9 engineering include sgRNA manipulation, PAM-recognition, specificity, deaminase fusions, reverse-transcriptase fusions, and structural rearrangements.

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