Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

McGrail, Maura
Wierson, Wesley
Simone, Brandon
WareJoncas, Zachary
Mann, Carla
Essner, Jeffrey
Welker, Jordan
Kar, Bibekananda
Emch, Michael
Friedberg, Iddo
Gendron, William
Barry, Michael
Clark, Karl
Dobbs, Drena
McGrail, Maura
Ekker, Stephen
Essner, Jeffrey
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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.

<p>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." <em>The CRISPR journal</em> 2, no. 6 (2019): 417-433. doi: <a href="https://doi.org/10.1089/crispr.2019.0026">10.1089/crispr.2019.0026</a>.</p>