Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase.

IF 44.7 1区综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES

Science Pub Date : 2025-05-15 DOI:10.1126/science.adt5199

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

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引用次数: 0

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.

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用实验室进化的crispr相关转座酶在人类细胞中插入可编程基因。

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（cast）催化rna引导的DNA整合，但迄今为止在人类细胞中表现出最小的活性。利用噬菌体辅助连续进化（PACE）技术，我们生成了整合活性平均提高200倍的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、用于免疫治疗的位点和与功能丧失疾病有关的基因）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，我们的发现建立了一个实验室进化的平台，并推进了一个在生命系统中可编程基因整合的通用系统。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Science 综合性期刊-综合性期刊

CiteScore

61.10

自引率

0.90%

发文量

审稿时长

2.1 months

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