Aug 2026· Microbiology Research· Vol 314, pp.
128697
· 0 citations· 73 references
Medicine
Abstract
Base editors (BEs) enable efficient A-to-G or C-to-T conversions without double-stranded DNA cleavage, but their editing windows remain difficult to tune, limiting genome engineering flexibility. Here, we engineered CRISPR/Cas12b sgRNA by introducing MS2 hairpins to recruit an MS2-N55K-cytidine deaminase-UGI complex, enabling programmable control of the editing window. Three modified sgRNAs were generated by replacing two loop regions, each producing distinct editing hotspots in E. coli. The AID*Δ-MSBE system (sgRNA1.1) generated a window near the PAM with peak activity at C7-C9, while the CDA-MSBE system (sgRNA1.2) produced a distal window with peak activity at C20-C23. Both systems exhibited identical editing patterns in Bacillus subtilis. A dual-orthogonal system (MS2 and PP7) was constructed to simultaneously recruit two deaminase complexes, restoring the classic dCas12b CBE editing pattern. Rifampicin resistance assays confirmed high targeting specificity with low off-target effects. As proof of concept, the MSBEs were successfully employed for the flexible reprogramming of sfGFP fluorescence and the targeted evolution of the endogenous gene rpsE, respectively. Collectively, we developed the MSBEs with tunable editing hotspots, providing innovative tools to enhance the flexibility and accessibility of BEs for genome engineering.
The effectiveness of the SaCas9 modular base editors, the robustness of the platform’s modularity, and its feasibility for convenient screening of target-specific base editors are demonstrated.
J. Collantes, Kellen Xu, M. Ruiz-Urigüen et al.· The CRISPR Journal· 0 citations
A single-nucleotide resolution ABE created by fusing a nickase Cas9, nCas9-H840A, with the deaminase domain of ADAR, which acts on DNA:RNA hybrids, instead of TadA, which acts on single-stranded DNA in conventional ABEs.
Hyeon Woo Im, Bada Jeong, Yeji Lee et al.· Nature Biotechnology· 1 citation
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
This review compares Cas9-mediated homology-directed repair (HDR) with generations of cytosine base editors (CBE1–CBE3), adenine base editors (ABE1-ABE7), and prime editors (PE1–PE3b), focusing on their mechanistic distinctions, efficiencies, delivery challenges, and therapeutic applications.
Anoushka Sinha· American Journal of Student...· 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
