Hodge Christine A, Donegan Niles P, Armstrong David A, Hayden Mathew S, Howell Alexandra L
Department of Dermatology, Dartmouth Health, Lebanon, NH, USA.
Department of Dermatology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA.
bioRxiv. 2025 Jul 11:2025.07.08.663680. doi: 10.1101/2025.07.08.663680.
Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. We evaluated CasX2 (Cas12e), a class II CRISPR system derived from , a non-pathogenic bacterium present in aquatic and terrestrial soils. CasX2 offers several advantages over Cas9 (Cas9) and Cas9 (Cas9), including its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and no known pre-existing immunity in humans. A recent study reported that a three amino acid substitution in CasX2 significantly enhanced cleavage activity (1). Therefore, we compared cleavage efficiency and double-stranded break repair characteristics between the native CasX2 and the variant, CasX2, for cleavage of , a gene that encodes the CCR5 receptor important for HIV-1 infection. Two CasX2 single guide RNAs (sgRNAs) were designed that flanked the 32 bases deleted in the natural Δ mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic In contrast, CasX2 using sgRNAs with 20 nt and 23 nt spacer lengths, enabled robust genomic cleavage of . Structural modeling indicated that two of the CasX2 substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2 in cellular gene excision. In sum, CasX2 consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.
新型CRISPR/Cas系统的开发增加了基因编辑治疗传染病、癌症和遗传疾病的机会。我们评估了CasX2（Cas12e），这是一种源自水生和陆地土壤中存在的非致病细菌的II类CRISPR系统。与Cas9和Cas9相比，CasX2具有几个优势，包括其较小的尺寸、独特的原间隔相邻基序（PAM）要求、促进同源定向修复的交错切割，以及在人类中不存在已知的预先存在的免疫。最近一项研究报告称，CasX2中的三个氨基酸取代显著增强了切割活性（1）。因此，我们比较了天然CasX2和变体CasX2在切割编码对HIV-1感染重要的CCR5受体的基因时的切割效率和双链断裂修复特征。设计了两个CasX2单向导RNA（sgRNA），它们位于天然Δ突变中缺失的32个碱基两侧。纳米孔测序表明，使用长度为17个核苷酸（nt）、20 nt或23 nt间隔序列的sgRNA的CasX2在切割基因组时无效。相比之下，使用20 nt和23 nt间隔长度的sgRNA的CasX2能够对进行强大的基因组切割。结构建模表明，CasX2的两个取代增强了sgRNA-DNA双链体稳定性，而第三个取代改善了催化位点内的DNA链排列。这些结构变化可能是CasX2在细胞基因切除中活性增加的基础。总之，在所有试验中，CasX2始终优于天然CasX2，代表了一种用于治疗应用的卓越基因编辑平台。
