A versatile platform for locus-scale genome rewriting and verification

Significance

Functional analysis of noncoding genomic regulatory elements, which harbor the majority of common human disease and trait associations, is complicated by their cellular and genomic context sensitivity. We developed Big-IN, a method for rewriting large segments of mammalian genomes, including full genes and their surrounding regulatory elements. We demonstrate a flexible genomic verification pipeline to identify correctly engineered cells. We expect Big-IN will enable technologies for synthesis and assembly of large DNAs to catalyze a synthetic approach to regulatory genomics.

Abstract

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.