title: Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.
abstract: Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. 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. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms.
authors.affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
authors.forename: Manuel
authors.initials: M
authors.lastname: Kaulich
authors.affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
authors.forename: Yeon J
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Lönn
authors.affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
authors.forename: Aaron D
authors.initials: AD
authors.lastname: Springer
authors.affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Meade
authors.affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA sdowdy@ucsd.edu.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Dowdy
mesh_terms: D000483:Alleles; D001483:Base Sequence; D016203:CDC2 Protein Kinase; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000235:genetics; D017931:DNA Primers; D000229:Dependovirus / Q000235:genetics*; D055785:Gene Knockdown Techniques; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D011995:Recombination, Genetic
chemical_list: D017931:DNA Primers; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases