利用实验室进化的CRISPR相关转座酶在人类细胞中进行可编程基因插入。
Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase.
作者信息
Witte Isaac P, Lampe George D, Eitzinger Simon, Miller Shannon M, Berríos Kiara N, McElroy Amber N, King Rebeca T, Stringham Olivia G, Gelsinger Diego R, Vo Phuc Leo H, Chen Albert T, Tolar Jakub, Osborn Mark J, Sternberg Samuel H, Liu David R
机构信息
Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
出版信息
Science. 2025 May 15;388(6748):eadt5199. doi: 10.1126/science.adt5199.
Programmable gene integration in human cells has the potential to enable mutation-agnostic treatments for loss-of-function genetic diseases and facilitate many applications in the life sciences. CRISPR-associated transposases (CASTs) catalyze RNA-guided DNA integration but thus far demonstrate minimal activity in human cells. Using phage-assisted continuous evolution (PACE), we generated CAST variants with >200-fold average improved integration activity. The evolved CAST system (evoCAST) achieves ~10 to 30% integration efficiencies of kilobase-size DNA cargoes in human cells across 14 tested genomic target sites, including safe harbor loci, sites used for immunotherapy, and genes implicated in loss-of-function diseases, with undetected indels and low levels of off-target integration. Collectively, our findings establish a platform for the laboratory evolution of CASTs and advance a versatile system for programmable gene integration in living systems.
人类细胞中的可编程基因整合有潜力实现针对功能丧失性遗传疾病的无突变治疗，并推动生命科学中的许多应用。CRISPR相关转座酶（CASTs）催化RNA引导的DNA整合，但迄今为止在人类细胞中表现出的活性极低。利用噬菌体辅助连续进化（PACE），我们生成了平均整合活性提高200倍以上的CAST变体。进化后的CAST系统（evoCAST）在14个测试的基因组靶位点（包括安全港位点、用于免疫治疗的位点以及与功能丧失性疾病相关的基因）上，在人类细胞中实现了约10%至30%的千碱基大小DNA货物整合效率，未检测到插入缺失且脱靶整合水平较低。总体而言，我们的研究结果建立了一个用于CASTs实验室进化的平台，并推进了一种用于活系统中可编程基因整合的通用系统。
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