Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.
Bottom Line: However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels.CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone.RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype.
Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.Show MeSH
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Mentions: The classical way to study mammalian gene function is to genetically knockout the gene or RNAi deplete the mRNA and hence, the protein of interest, to induce a phenotype. To confirm that the targeted gene was the causal gene requires rescuing the loss of function phenotype by ectopic expression of a variant of the same gene. Unfortunately, constitutive gene knockout can activate compensatory mechanisms that significantly impair the phenotype and conclusions to gene function (1,2). In contrast, acute RNAi-mediated gene depletion can reveal additional functions and lead to a more detailed molecular understanding (3). However, rescue of either gene deletion or depletion is equally fraught with potential pitfalls. Commonly used protein expression systems, including stably integrated genomic constructs under inducible promoters (4), often significantly overexpress the rescue protein, which alters the stoichiometry of protein–protein interactions and can lead to potential false-positive ‘rescue’ results (5,6). While the lack of proper gene regulation can potentially be addressed using minigenes or bacterial artificial chromosomes (6), these approaches represent an artificial situation for the cell. To close this gap, we developed a gene knockdown/rescue strategy that works at the endogenous gene level by combining RNAi selectivity with a highly efficient CRISPR–Cas (7–10) directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific phenotypic mutations of interest plus an allele-selective siRNA Sensitive (siSN) site (Figure 1).
Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.