PUBLICATION

Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

Authors
Wierson, W.A., Simone, B.W., WareJoncas, Z., Mann, C., Welker, J.M., Kar, B., Emch, M.J., Friedberg, I., Gendron, W.A.C., Barry, M.A., Clark, K.J., Dobbs, D.L., McGrail, M.A., Ekker, S.C., Essner, J.J.
ID
ZDB-PUB-191120-9
Date
2019
Source
The CRISPR journal   2(6): 417-433 (Journal)
Registered Authors
Clark, Karl, Ekker, Stephen C., Essner, Jeffrey, McGrail, Maura
Keywords
none
MeSH Terms
  • Animals
  • Base Sequence
  • CRISPR-Associated Proteins/genetics
  • CRISPR-Cas Systems/genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats/genetics*
  • DNA/chemistry
  • Gene Editing/methods*
  • Gene Targeting/methods
  • Genetic Engineering/methods
  • Genome/genetics
  • Humans
  • RNA/chemistry
  • RNA, Guide, Kinetoplastida/chemistry
  • Zebrafish/genetics
PubMed
31742435 Full text @ CRISPR J
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.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping