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

doi:10.1073/pnas.2023952118

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

Biomedical Engineering

Research output: Contribution to journal › Article › peer-review

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.

Original language	English (US)
Article number	e2023952118
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	118
Issue number	10
DOIs	https://doi.org/10.1073/pnas.2023952118
State	Published - Mar 9 2021

Keywords

Genome engineering
Genome writing
Regulatory genomics
Stem cells
Synthetic biology

ASJC Scopus subject areas

General

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10.1073/pnas.2023952118

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Brosh, R., Laurent, J. M., Ordoñez, R., Huang, E., Hogan, M. S., Hitchcock, A. M., Mitchell, L. A., Pinglay, S., Cadley, J. A., Luther, R. D., Truong, D. M., Boeke, J. D., & Maurano, M. T. (2021). A versatile platform for locus-scale genome rewriting and verification. Proceedings of the National Academy of Sciences of the United States of America, 118(10), Article e2023952118. https://doi.org/10.1073/pnas.2023952118

Brosh, R, Laurent, JM, Ordoñez, R, Huang, E, Hogan, MS, Hitchcock, AM, Mitchell, LA, Pinglay, S, Cadley, JA, Luther, RD, Truong, DM, Boeke, JD & Maurano, MT 2021, 'A versatile platform for locus-scale genome rewriting and verification', Proceedings of the National Academy of Sciences of the United States of America, vol. 118, no. 10, e2023952118. https://doi.org/10.1073/pnas.2023952118

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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.",

keywords = "Genome engineering, Genome writing, Regulatory genomics, Stem cells, Synthetic biology",

author = "Ran Brosh and Laurent, {Jon M.} and Raquel Ordo{\~n}ez and Emily Huang and Hogan, {Megan S.} and Hitchcock, {Angela M.} and Mitchell, {Leslie A.} and Sudarshan Pinglay and Cadley, {John A.} and Luther, {Raven D.} and Truong, {David M.} and Boeke, {Jef D.} and Maurano, {Matthew T.}",

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AU - Hitchcock, Angela M.

AU - Mitchell, Leslie A.

AU - Pinglay, Sudarshan

AU - Cadley, John A.

AU - Luther, Raven D.

AU - Truong, David M.

AU - Boeke, Jef D.

AU - Maurano, Matthew T.

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A versatile platform for locus-scale genome rewriting and verification

Abstract

Keywords

ASJC Scopus subject areas

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