Engineered novel high-fidelity Cas9 delivered as a ribonucleoprotein complex enables high frequency gene editing in human haematopoietic stem and progenitor cells

Translation of the CRISPR/Cas9 system to human therapeutics holds high promise.Specificity remains a concern, however, especially when modifying stem cellpopulations. We show that existing rationally-engineered Cas9 high fidelity variantshave reduced on-target activity using the therapeutically relevant ribonucleoprotein(RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolatevariants that retain activity in the RNP format. Introduction of a single point mutation,R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing. HiFiCas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci(HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem andprogenitor cells (HSPCs) as well as primary T-cells. We also show that the HiFi Cas9mediates high-level correction of the sickle cell disease (SCD)-causing Glu6Valmutation in SCD patient derived HSPCs. We anticipate that HiFi Cas9 will have wideutility for both basic science and therapeutic genome editing applications.

CRISPR/Cas9系统向人类治疗领域的转化具有极高应用前景。然而其特异性仍是亟待解决的核心问题，在对干细胞群体进行基因修饰时尤为突出。我们的研究发现，现有经理性设计的Cas9高保真变体，在采用治疗场景常用的核糖核蛋白（ribonucleoprotein, RNP）递送方式时，其靶上编辑活性有所降低。为此，我们开发了一种无偏倚细菌筛选策略，以分离出在RNP递送格式下仍保留活性的Cas9变体。通过引入单点突变R691A所获得的HiFi Cas9，既保持了较高的靶上编辑活性，又显著降低了脱靶编辑效应。HiFi Cas9可在人类CD34+造血干细胞和祖细胞（hematopoietic stem and progenitor cells, HSPCs）以及原代T细胞中，于五个治疗相关基因座（HBB、IL2RG、CCR5、HEXB、TRAC）实现高效的腺相关病毒6型（adeno-associated virus serotype 6, AAV6）介导的基因靶向编辑。我们还证实，HiFi Cas9可高效矫正源自镰状细胞病（sickle cell disease, SCD）患者的HSPCs中引发疾病的Glu6Val点突变。我们预计，HiFi Cas9将在基础科学研究及治疗性基因组编辑领域拥有广阔的应用前景。