Evaluating high-fidelity CRISPR-Cas nucleases in nucleosomal contexts using a quantitative framework

Designing Fidelity of CRISPR-Cas Endonucleases by Kinetic Insights

Quantitative profiling of intrinsic dCas9-DNA recognition reveals key determinants of guide RNA performance

CRISPR RNA architecture steers Cas9 catalysis and fidelity via a guide repeat clasp motif

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.

Cas-regulation-targeting chimera enables selective and tunable control of CRISPR/Cas12a

Abstract Selective and tunable regulation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a activity enables on-demand control, yet current strategies remain hindered by nonspecific regulation and limited tunability. Inspired by proximity effect, we present a Cas-regulation-targeting chimera (CasTAC) strategy that employs CRISPR RNA (crRNA) as a proximity mediator to carry phosphorothioate regulators to interfere with catalytic or recognition domains of Cas12a and consequently suppress its activity. This crRNA-induced proximity approach can effectively eliminate nonspecific interaction between phosphorothioate regulators and proteins within complex multi-enzyme systems, thereby enabling selective control over CRISPR/Cas12a activity. Furthermore, CRISPR/Cas12a activity can be finely tuned to different inhibitory levels by varying the number of phosphorothioate regulators. The CasTAC strategy also improves nuclease resistance and single-nucleotide discrimination, offering potential advances in the sensitivity of molecular diagnostics and the accuracy of gene editing. Notably, the CasTAC balances the kinetics of nucleic acid amplification and CRISPR cleavage, facilitating efficient product accumulation and resolving compatibility issues in one-pot assays. As a proof of concept, we develop a one-pot, one-step recombinase polymerase amplification–CasTAC assay that achieves over 1000-fold higher detection sensitivity than the conventional one-pot recombinase polymerase amplification−CRISPR/Cas12a assay. The CasTAC strategy provides a versatile framework for fine–tuning Cas activity and advances CRISPR technology toward refined and context-adaptable functionality.

CRISPR/Cas9-Based Genome Editing: Understanding Differences in DNA Repair Pathways, Profiles, and Outcomes

Over a decade of advances in Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-based technologies have culminated in the first-ever FDA-approved CRISPR/Cas-based therapy. Aside from this approved therapy for sickle cell anemia, several CRISPR/Cas-based therapies are currently under development or testing for a range of chronic diseases, including viral diseases like human immunodeficiency virus type 1 (HIV-1) infection, genetic diseases like familial hypercholesterolemia, and cancer. The success of these therapies hinges on the effective delivery of CRISPR/Cas9 components to target regions, efficient Cas endonuclease editing, repair profiles generated, and their resulting outcomes. Here, we discuss the factors that influence the generation of CRISPR/Cas9-generated repair edits, the overall profiles, and outcome prediction(s), as well as the analytical tools that have been developed to date. Finally, how this technology has been used towards a functional HIV-1 cure is discussed.

In vivo gene disruption and homology-directed repair in muscles and muscle stem cells using CRISPR/Cas9

Programmable endonucleases such as CRISPR/Cas9 provide powerful tools to edit mammalian genomes by engaging cellular mechanisms of DNA double-strand break (DSB) repair. CRISPR-catalysed homology-directed repair (CRISPR-HDR), though generally less efficient than other modes of DNA repair, holds particular promise to enable precise sequence replacement by targeted insertion of a homologous DNA template1,2. While recent studies have reported appreciable levels of HDR in cardiomyocytes in vivo3, skeletal muscle myofibres have historically been considered refractory to HDR-mediated genome editing4. Furthermore, how repair outcomes differ across tissues after systemic delivery of CRISPR/Cas9 editors, whether precise HDR editing can be achieved in regenerative tissue stem cells, and how developmental timing influences accessibility to CRISPR-induced repair remain unclear. Here, we use an adeno-associated virus (AAV)-delivered in vivo GFP-to-BFP colour-switching reporter system (AAV-GFP-to-BFP) to examine in vivo CRISPR-HDR with cellular- and tissue-level resolution. We find that postnatal cardiac muscle, skeletal muscle, and muscle stem cells undergo templated HDR at different rates across discrete developmental stages in mice. While HDR-edited muscle stem cells and myofibres were readily detectable after in vivo editing in juvenile mice, editing in neonatal mice yielded more efficient HDR in cardiac tissue. Based on these results, we adapted the CRISPR-HDR approach to rescue the therapeutically relevant Dmd mutation in mdx mice, demonstrating recoding to the wild-type protein sequence in both skeletal and cardiac muscles. These results provide a framework for advancing donor-templated DNA repair in living postnatal animals, and reveal unexpected cellular, developmental, and disease-related constraints on precise, therapeutic in vivo gene correction.