Natural variation in PAM recognition among SaCas9 orthologs is analyzed and StaCas9 is identified as a compact and efficient nuclease recognizing an NNG PAM, establishing StaCas9 as a high-performance genome-editing tool for therapeutic applications.
Five previously uncharacterized MG102-like Cas9d orthologs are identified that share the hallmark genomic, sequence, and structural features of type II-D Cas9 and establish compact MG102-like Cas9d orthologs as robust and specific genome editors and provide promising, single-AAV– compatible scaffolds for in vivo therapeutic genome editing.
Qiaochu Wang, Ahmed Saleh, G. S. Rao et al.· bioRxiv· 0 citations
An efficient Cas9d system (Cas9dUltra) is developed through gRNA and protein engineering, and its base editors (9dBEs) further developed through gRNA and protein engineering, enabling efficient and precise genome editing in human cells.
Qingquan Xiao, Zhijin Tian, Luqi Weng et al.· Advancement of science· 0 citations
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
Compact type II-C Cas9 nucleases are attractive for therapeutic genome editing because their small size enables packaging into adeno-associated viral (AAV) vectors, and their extended protospacer-adjacent motifs (PAMs) reduce off-target cleavage while expanding targeting scope. Yet characterized type II-C orthologs have edited mammalian cells far less efficiently than the canonical SpCas9. Here, we used embedding-based metagenomic mining of >4.7 × 10 proteins, combined with AlphaFold3 structure prediction and locus-context analysis, to identify three previously uncharacterized compact type II-C Cas9 orthologs, NsuCas9 (1,092 aa), PsuCas9 (1,084 aa), and GfoCas9 (1,074 aa), and benchmarked them in vitro and in human HEK293T cells. All three are robust RNA-guided nucleases with distinct PAM specificities (N CC, N NYAA, and N RHAA, respectively), divergent thermal profiles, and asymmetric sgRNA cross-compatibility. In human cells, PsuCas9 with an N ATAA PAM reaches 78.4% indels and matches or exceeds SpCas9 at multiple loci, representing the first natural compact type II-C ortholog reported to do so, while GfoCas9 and NsuCas9 add complementary coverage. All three show a strong deletion-biased repair signature and no detectable editing across 33 predicted off-target sites. These compact, high-fidelity nucleases expand the CRISPR targeting space for AAV-deliverable therapeutic editing.
Qiaochu Wang, Sivakrishna Rao Gundra, Rashid Aman et al.· bioRxiv· 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
Genomic manipulation has advanced from stochastic nuclease‐mediated disruption toward programmable, deterministic precision. Early clustered regularly interspaced short palindromic repeats (CRISPR) strategies enabled targeted mutagenesis through double‐strand breaks; however, their therapeutic application is limited by genotoxicity, chromosomal instability, and dependence on endogenous repair pathways that are difficult to predict. In this review, we examined the transition from gene editing to genome writing, an approach that decouples genomic modification from host repair pathways to better balance efficiency, precision, and payload delivery. We also discussed the principles of precision technologies, including base and prime editors, and described emerging large‐scale writers, such as CRISPR‐associated transposases and recombinase‐based bridge RNAs, which enable the integration of multi‐kilobase synthetic modules. Beyond enzymatic mechanisms, we further considered the combined use of generative artificial intelligence, structural biology, and novel delivery architectures as potential strategies to overcome current biological limitations. Taken together, these developments point toward Generative Biology, in which computational design and high‐throughput screening transform the genome from a static substrate into a more dynamic model for complex, synthetic functional design.