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Updated: Sep 15, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

Directed evolution expands CRISPR-Cas12a genome-editing capacity.

Enbo Ma^1,2, Kai Chen^1,2, Honglue Shi^1,3

¹Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, United States.

Nucleic Acids Research

|July 17, 2025

Summary

This summary is machine-generated.

Researchers engineered CRISPR-Cas12a (Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a) to recognize new DNA sequences. This enhanced genome editing tool, Flex-Cas12a, expands targeting capabilities for therapeutic and agricultural applications.

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CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

Published on: May 28, 2019

Area of Science:

Biotechnology
Molecular Biology
Genomics

Background:

CRISPR-Cas12a is a powerful RNA-guided genome editing system.
Its utility is limited by a narrow protospacer adjacent motif (PAM) recognition (5'-TTTV-3'), restricting targeting to ~1% of the genome.
Expanding PAM recognition is crucial for broader genomic applications.

Purpose of the Study:

To engineer variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition.
To overcome the targeting limitations of wild-type Cas12a.
To enhance the versatility of CRISPR-Cas12a for genome engineering.

Main Methods:

Bacterial-based directed evolution assay.
Rational protein engineering of Cas12a.
Biochemical and cell-based assays to characterize variants.
Analysis of PAM recognition specificity.

Main Results:

Identified Cas12a variants with expanded PAM recognition, including noncanonical motifs.
Developed Flex-Cas12a, which recognizes 5'-NYHV-3' PAMs in addition to the canonical 5'-TTTV-3' PAM.
Expanded DNA recognition sites to approximately 25% of the human genome.
Demonstrated retained recognition of the canonical PAM.

Conclusions:

Engineered Cas12a variants, particularly Flex-Cas12a, significantly broaden genome targeting capabilities.
Flex-Cas12a provides access to previously inaccessible genomic loci.
This advancement offers new opportunities for therapeutic and agricultural genome engineering.