小柯机器人

美国哈佛医学院Luca Pinello、Daniel E.Bauer和意大利维罗纳大学Rosalba Giugno组合作取得一项新突破。他们揭示了改变人类基因多样性基因编辑脱靶效率的预测方法。2022年12月15日出版的《自然-遗传学》杂志发表了这项成果。

研究人员开发了一种名为CRISPRme的有效工具，该工具考虑了单核苷酸多态性（SNP）和插入缺失遗传变异，以发现和优先考虑脱靶位点。研究人员通过靶向BCL11A增强子的指导RNA（gRNA）测试了该软件，在镰刀型细胞贫血病和β地中海贫血的临床试验中显现出应用前景，发现最常见的候选脱靶位点是在非洲血统人群中常见等位基因（MAF 4.5%)产生的，该等位基因引入了原间隔相邻基序（PAM）序列。

研究人员验证了SpCas9在CD34⁺造血干细胞和祖细胞（HSPC）中产生等位基因特异性插入缺失和倒置，虽然高保真Cas9能减弱这种脱靶。该研究说明了遗传变异为何作为基因编辑结果的修饰因子。研究人员预计变异脱靶评估将成为治疗性基因组编辑评估不可或缺的一部分，并为全面脱靶评估提供了一种强大的方法。

据介绍，CRISPR基因编辑在通过改变体细胞DNA序列进行疾病治疗方面具有很大前景。然而，预测脱靶概率的标准计算和生化方法则依赖于参考基因组。

附：英文原文

Title: Human genetic diversity alters off-target outcomes of therapeutic gene editing

Author: 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

Issue&Volume: 2022-12-15

Abstract: 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 single-nucleotide polymorphism (SNP) and indel genetic variants to nominate and prioritize off-target sites. We tested the software with a BCL11A enhancer targeting guide RNA (gRNA) 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+ hematopoietic stem and progenitor cells (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.

DOI: 10.1038/s41588-022-01257-y

Source: https://www.nature.com/articles/s41588-022-01257-y

Nature Genetics：《自然—遗传学》，创刊于1992年。隶属于施普林格·自然出版集团，最新IF：41.307
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投稿链接：https://mts-ng.nature.com/cgi-bin/main.plex