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Updated: Jun 10, 2025

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes
Xi Dawn Chen1,2,3, Zeyu Chen1,4,5, George Wythes1
1Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
A new tool called helicase-assisted continuous editing (HACE) enables targeted, long-range DNA mutations. This platform helps researchers understand gene function and discover new biological capabilities.
Area of Science:
- Genomics
- Molecular Biology
- Genetic Engineering
Background:
- Understanding sequence-function relationships in genomes is crucial but challenging.
- Current methods for targeted genomic mutation and evolution are limited in scope and application.
- Developing novel tools is essential for dissecting complex genetic mechanisms.
Purpose of the Study:
- To introduce a programmable platform for long-range, locus-specific genomic hypermutation.
- To demonstrate the utility of this platform in functional genomics studies.
- To enable the investigation of both coding and noncoding genetic elements.
Main Methods:
- Development of helicase-assisted continuous editing (HACE) platform.
- Utilizing CRISPR-Cas9 to guide a helicase-deaminase fusion enzyme.
- Application of HACE for targeted mutagenesis across large genomic intervals (>1000 bp).
Main Results:
- HACE successfully induced hypermutation at specific genomic loci.
- Identified mutations in MEK1 conferring kinase inhibitor resistance.
- Dissected the impact of variants on SF3B1-dependent missplicing.
- Evaluated noncoding variants within a CD69 immune enhancer.
Conclusions:
- HACE is a powerful tool for investigating coding and noncoding variants.
- The platform facilitates the uncovering of combinatorial sequence-to-function relationships.
- HACE enables the directed evolution of novel biological functions.
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