Human genetic diversity alters off-target outcomes of therapeutic gene editing.
Human genetic diversity alters off-target outcomes of therapeutic gene editing.
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DOI：
10.1038/s41588-022-01257-y
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发表时间：
2023-01
期刊：
影响因子：
30.8
通讯作者：
Pinello, Luca
中科院分区：
文献类型：
--
作者：
Cancellieri, Samuele;Zeng, Jing;Lin, Linda Yingqi;Tognon, Manuel;Nguyen, My Anh;Lin, Jiecong;Bombieri, Nicola;Maitland, Stacy A.;Ciuculescu, Marioara-Felicia;Katta, Varun;Tsai, Shengdar Q.;Armant, Myriam;Wolfe, Scot A.;Giugno, Rosalba;Bauer, Daniel E.;Pinello, Luca
CRISPR基因编辑有望修改体细胞中的DNA序列以治疗疾病。然而，预测脱靶潜力的标准计算和生物化学方法集中于参考基因组。我们开发了一种名为CRISPRme的有效工具，该工具考虑SNP和indel遗传变异来提名和优先考虑脱靶位点。我们用BCL11A增强子靶向向导RNA测试了该软件，该软件在镰状细胞病和β地中海贫血的临床试验中显示出希望，并发现最佳候选脱靶是由非洲血统人群中常见的等位基因（MAF 4.5%）产生的，该等位基因引入了原型间隔区邻近基序（PAM）序列。我们验证了SpCas9在CD34+ HSPC中产生严格的等位基因特异性插入缺失和臂间倒位，尽管高保真Cas9减轻了这种脱靶。本报告说明了遗传变异应如何被视为基因编辑结果的修饰符。我们预计，变体感知脱靶评估将成为治疗性基因组编辑评估的组成部分，并为全面的脱靶提名提供强有力的方法。
CRISPR gene editing holds great promise to modify DNA sequences in somatic cells to treat disease. However, standard computational and biochemical methods to predict off-target potential focus on reference genomes. We developed an efficient tool called CRISPRme that considers SNP and indel genetic variants to nominate and prioritize off-target sites. We tested the software with a BCL11A enhancer targeting guide RNA showing promise in clinical trials for sickle cell disease and β-thalassemia and found that the top candidate off-target is produced by an allele common in African-ancestry populations (MAF 4.5%) that introduces a protospacer adjacent motif (PAM) sequence. We validated that SpCas9 generates strictly allele-specific indels and pericentric inversions in CD34+ HSPCs, although high-fidelity Cas9 mitigates this off-target. This report illustrates how genetic variants should be considered as modifiers of gene editing outcomes. We expect that variant-aware off-target assessment will become integral to therapeutic genome editing evaluation and provide a powerful approach for comprehensive off-target nomination.
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