使用噬菌体辅助连续进化（PACE），该研究组生成了具有200倍平均提高整合活性的CAST变体。进化的CAST系统（evoCAST）在人类细胞中通过14个测试的基因组靶点（包括安全港位点、免疫治疗主题位点和与功能丧失疾病相关的基因）实现了千碱基大小的DNA货物的10%至30%的整合效率，这些靶点具有未检测到的缺失和低水平的脱靶整合。总的来说，他们的发现为铸模的实验室进化建立了一个平台，并推进了一个在生命系统中可编程基因整合的通用系统。
据了解，人类细胞中的可编程基因整合有可能实现功能丧失遗传疾病的突变不可知论治疗，并促进在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，但它们在人类细胞中表现出最小的活性。
附：英文原文
Title: Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase
Author: Isaac P. Witte, George D. Lampe, Simon Eitzinger, Shannon M. Miller, Kiara N. Berríos, Amber N. McElroy, Rebeca T. King, Olivia G. Stringham, Diego R. Gelsinger, Phuc Leo H. Vo, Albert T. Chen, Jakub Tolar, Mark J. Osborn, Samuel H. Sternberg, David R. Liu
Issue&Volume: 2025-05-15
Abstract: 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.
DOI: adt5199
Source: https://www.science.org/doi/10.1126/science.adt5199