Posted in BAM, biotech, cas9, crispr, DEL, DNA, electrotransfection, GEO, indel, mol, pymol, R, RMA, RNA, SED

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

 August 26, 2021



doi: 10.1016/j.mex.2021.101419.


eCollection 2021.

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Anil K Challa et al.


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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.

Conflict of interest statement

The Authors confirm that there are no conflicts of interest.

Figures


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Graphical abstract


Fig. 1



Fig. 1

Assessing nuclease activity of sgRNA-Cas9 RNP complex in electroporated rat embryos. (A) Schematic of the genomic location along with CRISPR targets (blue arrows C1, C2) and PCR primers (F1, R1). (B, C) Schematics of the sequence at the exact target sites in the context of the wildtype (B) and modified (C) alleles. (D-F) Inverted images from 6% TBE-PAGE analysis of (D) PCR product of the wildtype allele. (E) HMA indicating the presence of heteroduplexes in PCR products obtained from zygotes electroporated with either guide C1 or C2. (F) RFLP analysis of the same PCR product with XhoI shows digested products indicated by white asterisks.

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