Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells
Date
Authors
Major Professor
Advisor
Committee Member
Journal Title
Journal ISSN
Volume Title
Publisher
Authors
Research Projects
Organizational Units
The Department of Genetics, Development, and Cell Biology seeks to teach subcellular and cellular processes, genome dynamics, cell structure and function, and molecular mechanisms of development, in so doing offering a Major in Biology and a Major in Genetics.
History
The Department of Genetics, Development, and Cell Biology was founded in 2005.
Related Units
- College of Agriculture and Life Sciences (parent college)
- College of Liberal Arts and Sciences (parent college)
Journal Issue
Is Version Of
Versions
Series
Department
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.