Campus Units

Genetics, Development and Cell Biology, Veterinary Microbiology and Preventive Medicine

Document Type

Article

Publication Version

Published Version

Publication Date

12-2019

Journal or Book Title

The CRISPR Journal

Volume

2

Issue

6

First Page

417

Last Page

433

DOI

10.1089/crispr.2019.0026

Abstract

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

Comments

This article is published as Wierson, Wesley A., Brandon W. Simone, Zachary WareJoncas, Carla Mann, Jordan M. Welker, Bibekananda Kar, Michael J. Emch et al. "Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells." The CRISPR journal 2, no. 6 (2019): 417-433. doi: 10.1089/crispr.2019.0026.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

Copyright Owner

Wesley A. Wierson et al.

Language

en

File Format

application/pdf

Share

COinS