PDF(Pubmed)
Abstract：
Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity and without observed off-target editing. The main protocol modifications needed to achieve such high efficiencies were the addition of the DNA-PK inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in the donor in addition to mutations disrupting the PAM sequence. Critically, editing was even across the progenitor hierarchy, did not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays or the frequency of high self-renewal potential long-term culture initiating cells. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donors. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.
摘要：
原发性造血干细胞和祖细胞（HSPC）中的精确基因编辑将促进单基因疾病的治愈性治疗以及疾病建模。即使使用CRISPR/Cas系统，也能实现精确的效率，然而,保持有限。通过引导RNA递送的优化，供体设计,和添加剂，我们现在已经获得了初级脐带血HSCPs的平均精确编辑效率>90%,且毒性最小,且未观察到脱靶编辑.实现这种高效率所需的主要方案修改是添加DNA-PK抑制剂AZD7648,以及除了破坏PAM序列的突变之外,在供体中包含间隔区破坏沉默突变。严重的,编辑甚至跨越了祖先阶层，在集落形成细胞测定或高自我更新潜力的长期培养起始细胞的频率中，不会实质上扭曲层次结构或影响谱系输出。由于许多疾病的建模需要杂合性，我们还证明,通过添加确定的突变体和野生型供体的混合物,可以调整整体编辑和接合性.有了这些优化，编辑在接近完美的效率现在可以直接在人类HSPC完成。这将为治疗策略和疾病建模开辟新的途径。
