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Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells
Wesley A Wierson1, Brandon W Simone2, Zachary WareJoncas2
1Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa.
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.
Insights
CRISPR-ErCas12a gene editing in zebrafish and human cells shows more efficient DNA repair than CRISPR-Cas9. This novel CRISPR-Cas system enhances genome engineering flexibility for researchers.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas systems enable targeted DNA double-strand breaks using RNA-guided effector proteins.
- Effector proteins exhibit variations in DNA binding, nuclease activity, and target site recognition.
- ErCas12a, a Cas12a family member from Eubacterium rectale, represents a novel tool for genome engineering.
Purpose of the Study:
- To characterize the activity of ErCas12a in zebrafish and human cells.
- To compare the DNA repair efficiency of CRISPR-ErCas12a with CRISPR-Cas9.
- To demonstrate the application of CRISPR-ErCas12a for gene integration using the GeneWeld method.
Main Methods:
- Utilized a fluorescent reporter system to assess CRISPR-ErCas12a activity.
- Employed the GeneWeld gene targeting method for integrating reporter alleles.
- Performed experiments in both zebrafish and human cell lines.
Main Results:
- CRISPR-ErCas12a demonstrated more efficient strand annealing mediated DNA repair compared to CRISPR-Cas9.
- Successfully integrated reporter alleles into the genomes of zebrafish and human cells using CRISPR-ErCas12a and GeneWeld.
- Characterized ErCas12a activity in vivo in zebrafish and in vitro in human cells.
Conclusions:
- CRISPR-ErCas12a is an effective tool for genome engineering in diverse model systems.
- This study expands the CRISPR-Cas toolbox, offering researchers greater flexibility.
- ErCas12a provides an alternative for applications requiring efficient strand annealing mediated DNA repair.
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