Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes

Xi Dawn Chen; Zeyu Chen; George Wythes; Yifan Zhang; Benno C Orr; Gary Sun; Yu-Kai Chao; Andrea Navarro Torres; Ka Thao; Mounica Vallurupalli; Jing Sun; Mehdi Borji; Emre Tkacik; Haiqi Chen; Bradley E Bernstein; Fei Chen

doi:10.1126/science.adn5876

Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes

Science. 2024 Oct 11;386(6718):eadn5876. doi: 10.1126/science.adn5876. Epub 2024 Oct 11.

Authors

Xi Dawn Chen^#^{1

2

3}, Zeyu Chen^#^{1

4

5}, George Wythes¹, Yifan Zhang¹, Benno C Orr^{1

2}, Gary Sun^{4

5}, Yu-Kai Chao^{1

6}, Andrea Navarro Torres¹, Ka Thao⁴, Mounica Vallurupalli⁷, Jing Sun¹, Mehdi Borji¹, Emre Tkacik³, Haiqi Chen^{8

9}, Bradley E Bernstein^{1

4

5

10}, Fei Chen^{1

2}

Affiliations

¹ Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
² Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
³ Systems, Synthetic, and Quantitative Biology PhD Program, Harvard University, Cambridge, MA 02138, USA.
⁴ Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
⁵ Department of Cell Biology and Pathology, Harvard Medical School, Boston, MA 02115, USA.
⁶ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
⁷ Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁸ Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁹ Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹⁰ The Novo Nordisk Foundation Center for Genomic Mechanisms of Disease, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

^# Contributed equally.

PMID: 39388570
DOI: 10.1126/science.adn5876

Abstract

Deciphering the context-specific relationship between sequence and function is a major challenge in genomics. Existing tools for inducing locus-specific hypermutation and evolution in the native genome context are limited. Here we present a programmable platform for long-range, locus-specific hypermutation called helicase-assisted continuous editing (HACE). HACE leverages CRISPR-Cas9 to target a processive helicase-deaminase fusion that incurs mutations across large (>1000-base pair) genomic intervals. We applied HACE to identify mutations in mitogen-activated protein kinase kinase 1 (MEK1) that confer kinase inhibitor resistance, to dissect the impact of individual variants in splicing factor 3B subunit 1 (SF3B1)-dependent missplicing, and to evaluate noncoding variants in a stimulation-dependent immune enhancer of CD69. HACE provides a powerful tool for investigating coding and noncoding variants, uncovering combinatorial sequence-to-function relationships, and evolving new biological functions.

MeSH terms

CRISPR-Cas Systems*
DNA Helicases* / chemistry
DNA Helicases* / genetics
Gene Editing* / methods
HEK293 Cells
Humans
MAP Kinase Kinase 1 / genetics
Mutagenesis*
Mutation
RNA Splicing

Substances

DNA Helicases
MAP Kinase Kinase 1