A versatile platform for locus-scale genome rewriting and verification

Ran Brosh, Jon M. Laurent, Raquel Ordoñez, Emily Huang, Megan S. Hogan, Angela M. Hitchcock, Leslie A. Mitchell, Sudarshan Pinglay, John A. Cadley, Raven D. Luther, David M. Truong, Jef D. Boeke, Matthew T. Maurano

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article numbere2023952118
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number10
StatePublished - Mar 9 2021


  • Genome engineering
  • Genome writing
  • Regulatory genomics
  • Stem cells
  • Synthetic biology

ASJC Scopus subject areas

  • General


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