Validation of gene editing efficiency with CRISPR-Cas9 system directly in rat zygotes using electroporation mediated delivery and embryo culture

Anil K Challa; Denise Stanford; Antonio Allen; Lawrence Rasmussen; Ferdinand K Amanor; S Vamsee Raju

doi:10.1016/j.mex.2021.101419

Validation of gene editing efficiency with CRISPR-Cas9 system directly in rat zygotes using electroporation mediated delivery and embryo culture

MethodsX. 2021 Jun 18:8:101419. doi: 10.1016/j.mex.2021.101419. eCollection 2021.

Authors

Anil K Challa¹, Denise Stanford², Antonio Allen², Lawrence Rasmussen², Ferdinand K Amanor², S Vamsee Raju²

Affiliations

¹ Department of Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
² Cystic Fibrosis Research Center, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.

Abstract

Successful use of the CRISPR-Cas9 system for gene manipulation relies on identifying effective and efficient guide RNA sequences (gRNAs). When the goal is to create transgenic animal/rodent models by knocking-in desired sequences using homology-directed repair (HDR), selecting effective guides becomes even more critical to minimize developmental time and resources. Currently, validation experiments for gRNAs for generating rat models are carried out using immortalized rat cells. However, there are several limitations with using such cell lines, including ploidy of the genome, non-predictive transfection efficiency, and the ability to identify gene modifications efficiently within diverse cell populations. Since embryos are authentic representatives of live animals compared to cell lines, validating CRISPR guides for their nuclease activity in freshly isolated embryos will provide greater accuracy of in vivo gene editing efficiency. In contrast to microinjections, delivery by electroporation is a more accessible method that can be simple and does not require special skills and equipment. We demonstrate an accessible workflow to either delete or edit target genes in vivo in rats using the efficient editing of a human mutation in alpha7 nicotinic acetylcholine receptor subunit (CHRNA7) ortholog using electroporation as a delivery method for CRISPR-Cas9 ribonucleoprotein complexes in rat embryos.•Upon identifying CRISPR targets at the desired genetic alteration site, we designed homologydriven repair (HDR) templates for effective and easy identification of gene editing by Restriction Fragment Length Polymorphism (RFLP).•Cultured rat embryos can be genotyped to assess CRISPR activity as seen by either presence of indels resulting from NHEJ or knock-in of repair template resulting from homology driven repair.•Heteroduplex mobility assay (HMA) and Restriction Fragment Length Polymorphism (RFLP) of PCR products can be performed reliably and reproducibly at a low-cost.

Keywords: Chrna7; Heteroduplex mobility assay (HMA); Homology-driven repair (HDR); Indels.

Grants and funding

R01 AA027528/AA/NIAAA NIH HHS/United States