Routine rewriting of loci associated with human traits and diseases would facilitate their functional analysis. However, existing DNA integration approaches are limited in terms of scalability and portability across genomic loci and cellular contexts. We describe Big-IN, a versatile platform for targeted integration of large DNAs into mammalian cells. CRISPR/Cas9-mediated targeting of a landing pad enables subsequent recombinase-mediated delivery of variant payloads and efficient positive/negative selection for correct clones in mammalian stem cells. We demonstrate integration of constructs up to 143 kb, and an approach for one-step scarless delivery. We developed a staged pipeline combining PCR genotyping and targeted capture sequencing for economical and comprehensive verification of engineered stem cells. Our approach should enable combinatorial interrogation of genomic functional elements and systematic locus-scale analysis of genome function.

对与人类特征和疾病相关的基因座进行常规重写将有助于其功能分析。然而，现有的 DNA 整合方法在跨基因组位点和细胞环境的可扩展性和可移植性方面受到限制。我们描述了 Big-IN，这是一个多功能平台，用于将大 DNA 靶向整合到哺乳动物细胞中。CRISPR/Cas9 介导的着陆垫靶向使随后的重组酶介导的变异有效载荷的传递和哺乳动物干细胞中正确克隆的有效正/负选择成为可能。我们展示了长达 143 kb 的构建体的集成，以及一种一步无痕交付的方法。我们开发了结合 PCR 基因分型和靶向捕获测序的分阶段管道，用于对工程干细胞进行经济和全面的验证。