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Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Kaulich M, Lee YJ, Lönn P, Springer AD, Meade BR, Dowdy SF - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

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Allele specific RNAi depletion between Cdk2-WT and Cdk2-T160A genotypes. (A) Genomic PCR analysis on clones obtained with control Cdk2-SN-T160A rAAV infection alone showing 1/19 (5%) recombination events. (B) Genomic PCR analysis on clones obtained with CRISPR-gCdk2 plus Cdk2-SN-T160A rAAV with 44/48 (92%) recombination events. (C) Bar graph of recombination efficiency of combining CRISPR-mediated DNA-cleavage with rAAV-mediated template delivery analyzed by genomic PCR. (D) Allele-specific sequencing results of wild type allele and recombined Cdk2-T160A allele. (E) Cdk2 immunoblot analysis of Cdk2+/SN and Cdk2+/SN-T160A cells transfected with control siCtrl, siWT, siSN or both, as indicated. The location of the T160-PO4 Cdk2 protein is indicated. (F) Propidium iodide DNA flow cytometry cell cycle analysis of Cdk2+/SN-T160A cells transfected with either siSN, which depletes Cdk2-T160A and expresses wild type Cdk2 (no phenotype), or siWT, which depletes wild type Cdk2 and expresses Cdk2-T160A, resulting in a cell cycle arrest phenotype. Data is from three independent experiments; error bars represent standard deviation.
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Figure 3: Allele specific RNAi depletion between Cdk2-WT and Cdk2-T160A genotypes. (A) Genomic PCR analysis on clones obtained with control Cdk2-SN-T160A rAAV infection alone showing 1/19 (5%) recombination events. (B) Genomic PCR analysis on clones obtained with CRISPR-gCdk2 plus Cdk2-SN-T160A rAAV with 44/48 (92%) recombination events. (C) Bar graph of recombination efficiency of combining CRISPR-mediated DNA-cleavage with rAAV-mediated template delivery analyzed by genomic PCR. (D) Allele-specific sequencing results of wild type allele and recombined Cdk2-T160A allele. (E) Cdk2 immunoblot analysis of Cdk2+/SN and Cdk2+/SN-T160A cells transfected with control siCtrl, siWT, siSN or both, as indicated. The location of the T160-PO4 Cdk2 protein is indicated. (F) Propidium iodide DNA flow cytometry cell cycle analysis of Cdk2+/SN-T160A cells transfected with either siSN, which depletes Cdk2-T160A and expresses wild type Cdk2 (no phenotype), or siWT, which depletes wild type Cdk2 and expresses Cdk2-T160A, resulting in a cell cycle arrest phenotype. Data is from three independent experiments; error bars represent standard deviation.

Mentions: To investigate gene function at endogenous gene expression and regulation levels using CRISPR plus rAAV, we introduced an Ala mutation into Cdk2′s activating T-loop phosphorylation site (T160A) in exon 4 and placed the siSN sequence in exon 2 (Figure 1B and C). Based on locus and integration-specific PCR, infection of RPE cells with only Cdk2-SN-T160A-NeoR rAAV (no CRIPSR) resulted in 1/19 (5%) NeoR colonies containing recombined Cdk2-SN-T160A single alleles, and no double alleles (Figure 3A) (Supplementary Figure S2I). In contrast, infection of Cdk2-SN-T160A-NeoR rAAV plus transfection of pX330 CRISPR/Cas9-gCdk2 plasmid resulted in an impressive 44/48 (92%) NeoR colonies containing recombined Cdk2-SN-T160A (Figure 3B). Of the 44 Cdk2-SN-T160A colonies, 41 (93%) were single Cdk2-SN-T160A alleles (Cdk2+/SN-T160A) and 3 (7%) were double alleles (Cdk2SN-T160A/SN-T160A) (Figure 3C) (Supplementary Figure S2J). We sequenced 4 clones chosen at random and all four contained both the siSN sequence and the T160A mutation (Figure 3D). Similarly to the Cdk2 results, recombination of the Cdk1 gene with Cdk1-SN-T161E-NeoR rAAV plus CRISPR/Cas9-gCdk1 plasmid resulted in 100% recombined clones (40/40) (Supplementary Figure S5). However, we note that unlike Cdk2 where Cdk1 can partially compensate for complete loss of Cdk2 in Cdk2SN-T160A/SN-T160A clones, Cdk2 cannot compensate for Cdk1 loss and we found no double Cdk1SN-T161E/SN-T161E clones. Transfection of either control siCtrl, siWT, siSN or both siWT and siSN into control Cdk2+/SN resulted in allelic-specific depletion of Cdk2 (Figure 3E). Transfection of siWT into Cdk2+/SN-T160A cells resulted in loss of the wild type, active Cdk2 T160 phosphorylated version (Figure 3E), which was accompanied by a characteristic phenotypic cell cycle arrest (Figure 3F), with retention of the inactive Cdk2SN-T160A allele. In contrast, transfection of siSN, resulted in the selective loss of the Cdk2SN-T160A allele with continued expression of the wild type Cdk2 allele and had no effect on cell cycle progression or Cdk2 T160 phosphorylation. Taken 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 with the gene of interest expressed and regulated under endogenous conditions.


Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Kaulich M, Lee YJ, Lönn P, Springer AD, Meade BR, Dowdy SF - Nucleic Acids Res. (2015)

Allele specific RNAi depletion between Cdk2-WT and Cdk2-T160A genotypes. (A) Genomic PCR analysis on clones obtained with control Cdk2-SN-T160A rAAV infection alone showing 1/19 (5%) recombination events. (B) Genomic PCR analysis on clones obtained with CRISPR-gCdk2 plus Cdk2-SN-T160A rAAV with 44/48 (92%) recombination events. (C) Bar graph of recombination efficiency of combining CRISPR-mediated DNA-cleavage with rAAV-mediated template delivery analyzed by genomic PCR. (D) Allele-specific sequencing results of wild type allele and recombined Cdk2-T160A allele. (E) Cdk2 immunoblot analysis of Cdk2+/SN and Cdk2+/SN-T160A cells transfected with control siCtrl, siWT, siSN or both, as indicated. The location of the T160-PO4 Cdk2 protein is indicated. (F) Propidium iodide DNA flow cytometry cell cycle analysis of Cdk2+/SN-T160A cells transfected with either siSN, which depletes Cdk2-T160A and expresses wild type Cdk2 (no phenotype), or siWT, which depletes wild type Cdk2 and expresses Cdk2-T160A, resulting in a cell cycle arrest phenotype. Data is from three independent experiments; error bars represent standard deviation.
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Figure 3: Allele specific RNAi depletion between Cdk2-WT and Cdk2-T160A genotypes. (A) Genomic PCR analysis on clones obtained with control Cdk2-SN-T160A rAAV infection alone showing 1/19 (5%) recombination events. (B) Genomic PCR analysis on clones obtained with CRISPR-gCdk2 plus Cdk2-SN-T160A rAAV with 44/48 (92%) recombination events. (C) Bar graph of recombination efficiency of combining CRISPR-mediated DNA-cleavage with rAAV-mediated template delivery analyzed by genomic PCR. (D) Allele-specific sequencing results of wild type allele and recombined Cdk2-T160A allele. (E) Cdk2 immunoblot analysis of Cdk2+/SN and Cdk2+/SN-T160A cells transfected with control siCtrl, siWT, siSN or both, as indicated. The location of the T160-PO4 Cdk2 protein is indicated. (F) Propidium iodide DNA flow cytometry cell cycle analysis of Cdk2+/SN-T160A cells transfected with either siSN, which depletes Cdk2-T160A and expresses wild type Cdk2 (no phenotype), or siWT, which depletes wild type Cdk2 and expresses Cdk2-T160A, resulting in a cell cycle arrest phenotype. Data is from three independent experiments; error bars represent standard deviation.
Mentions: To investigate gene function at endogenous gene expression and regulation levels using CRISPR plus rAAV, we introduced an Ala mutation into Cdk2′s activating T-loop phosphorylation site (T160A) in exon 4 and placed the siSN sequence in exon 2 (Figure 1B and C). Based on locus and integration-specific PCR, infection of RPE cells with only Cdk2-SN-T160A-NeoR rAAV (no CRIPSR) resulted in 1/19 (5%) NeoR colonies containing recombined Cdk2-SN-T160A single alleles, and no double alleles (Figure 3A) (Supplementary Figure S2I). In contrast, infection of Cdk2-SN-T160A-NeoR rAAV plus transfection of pX330 CRISPR/Cas9-gCdk2 plasmid resulted in an impressive 44/48 (92%) NeoR colonies containing recombined Cdk2-SN-T160A (Figure 3B). Of the 44 Cdk2-SN-T160A colonies, 41 (93%) were single Cdk2-SN-T160A alleles (Cdk2+/SN-T160A) and 3 (7%) were double alleles (Cdk2SN-T160A/SN-T160A) (Figure 3C) (Supplementary Figure S2J). We sequenced 4 clones chosen at random and all four contained both the siSN sequence and the T160A mutation (Figure 3D). Similarly to the Cdk2 results, recombination of the Cdk1 gene with Cdk1-SN-T161E-NeoR rAAV plus CRISPR/Cas9-gCdk1 plasmid resulted in 100% recombined clones (40/40) (Supplementary Figure S5). However, we note that unlike Cdk2 where Cdk1 can partially compensate for complete loss of Cdk2 in Cdk2SN-T160A/SN-T160A clones, Cdk2 cannot compensate for Cdk1 loss and we found no double Cdk1SN-T161E/SN-T161E clones. Transfection of either control siCtrl, siWT, siSN or both siWT and siSN into control Cdk2+/SN resulted in allelic-specific depletion of Cdk2 (Figure 3E). Transfection of siWT into Cdk2+/SN-T160A cells resulted in loss of the wild type, active Cdk2 T160 phosphorylated version (Figure 3E), which was accompanied by a characteristic phenotypic cell cycle arrest (Figure 3F), with retention of the inactive Cdk2SN-T160A allele. In contrast, transfection of siSN, resulted in the selective loss of the Cdk2SN-T160A allele with continued expression of the wild type Cdk2 allele and had no effect on cell cycle progression or Cdk2 T160 phosphorylation. Taken 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 with the gene of interest expressed and regulated under endogenous conditions.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

Show MeSH
Related in: MedlinePlus