Enhanced cleavage of genomic CCR5 using CasX2Max

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2Max, a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural CCR5 ∆32 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 CCR5. In contrast, CasX2Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic CCR5. Structural modelling indicated that two of the CasX2Max amino acid 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 CasX2Max in cellular gene excision. In sum, CasX2Max 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，为利用基因编辑技术治疗传染病、癌症及遗传疾病拓展了机遇。CasX2（PlmCas12e）属于源自浮霉菌门（Planctomycetes）的II类CRISPR系统，该菌为一种存在于水生及陆生土壤中的非致病性细菌。相较于酿脓链球菌Cas9（Streptococcus pyogenes Cas9, SpCas9）与金黄色葡萄球菌Cas9（Staphylococcus aureus Cas9, SaCas9），CasX2作为潜在治疗用CRISPR系统具备多项优势：其分子尺寸更小、具有独特的原间隔序列邻近基序（protospacer adjacent motif, PAM）识别要求、可产生交错切割以促进同源定向修复，且在人体中不存在预存免疫反应。本研究针对CCR5基因——该基因编码对HIV-1感染至关重要的CCR5受体——设计靶向靶点，比较了CasX2与新近构建的含3个氨基酸替换的CasX2变体CasX2Max的切割效率及双链断裂修复特性。研究设计了两条单引导RNA（single guide RNA, sgRNAs），其结合位点位于天然CCR5 ∆32突变缺失的32个碱基侧翼区域。纳米孔测序结果显示，当使用间隔序列长度为17 nt、20 nt或23 nt的sgRNA时，CasX2无法有效切割基因组CCR5位点。与之形成对比的是，使用间隔序列长度为20 nt与23 nt的sgRNA时，CasX2Max可实现基因组CCR5位点的有效切割。结构建模分析表明，CasX2Max的3个氨基酸替换中，有2个提升了sgRNA-DNA双链的稳定性，第三个则优化了催化位点内的DNA链排列。这些结构变化可能是CasX2Max在细胞基因切除实验中活性提升的分子基础。综上，在所有实验检测中，CasX2Max的表现均优于野生型CasX2，是一款更具应用前景的治疗性基因编辑平台。