Limits...
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.

Show MeSH

Related in: MedlinePlus

CRISPR/Cas9 plus rAAV targeted recombination. (A) Study concept of combining CRISPR/Cas9 plasmid co-expressing a specific guide strand with rAAV. (B) Design of rAAV Cdk2 promoter-less construct. (C) Graphic depiction of the rAAV-targeting strategy to introduce siSN silent point-mutations into exon 2 and a point mutation at Thr-160 to Ala (T160A) of human Cdk2. Highlighted are the two crucial steps of ‘targeting’ (Cas9 + gRNA + ssDNA) and allele ‘restoration’ (+Cre). Note locations of gRNA, siSN and T160A. (D) Introduction of siSN sequence plus a gene mutation into one allele of a given gene allows for selective depletion of the wild type allele by siWT resulting in a mutant phenotype, selective depletion of the mutant gene by siSN resulting in a rescued phenotype or depletion of both alleles to mimic a loss of function phenotype.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4402508&req=5

Figure 1: CRISPR/Cas9 plus rAAV targeted recombination. (A) Study concept of combining CRISPR/Cas9 plasmid co-expressing a specific guide strand with rAAV. (B) Design of rAAV Cdk2 promoter-less construct. (C) Graphic depiction of the rAAV-targeting strategy to introduce siSN silent point-mutations into exon 2 and a point mutation at Thr-160 to Ala (T160A) of human Cdk2. Highlighted are the two crucial steps of ‘targeting’ (Cas9 + gRNA + ssDNA) and allele ‘restoration’ (+Cre). Note locations of gRNA, siSN and T160A. (D) Introduction of siSN sequence plus a gene mutation into one allele of a given gene allows for selective depletion of the wild type allele by siWT resulting in a mutant phenotype, selective depletion of the mutant gene by siSN resulting in a rescued phenotype or depletion of both alleles to mimic a loss of function phenotype.

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).


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)

CRISPR/Cas9 plus rAAV targeted recombination. (A) Study concept of combining CRISPR/Cas9 plasmid co-expressing a specific guide strand with rAAV. (B) Design of rAAV Cdk2 promoter-less construct. (C) Graphic depiction of the rAAV-targeting strategy to introduce siSN silent point-mutations into exon 2 and a point mutation at Thr-160 to Ala (T160A) of human Cdk2. Highlighted are the two crucial steps of ‘targeting’ (Cas9 + gRNA + ssDNA) and allele ‘restoration’ (+Cre). Note locations of gRNA, siSN and T160A. (D) Introduction of siSN sequence plus a gene mutation into one allele of a given gene allows for selective depletion of the wild type allele by siWT resulting in a mutant phenotype, selective depletion of the mutant gene by siSN resulting in a rescued phenotype or depletion of both alleles to mimic a loss of function phenotype.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402508&req=5

Figure 1: CRISPR/Cas9 plus rAAV targeted recombination. (A) Study concept of combining CRISPR/Cas9 plasmid co-expressing a specific guide strand with rAAV. (B) Design of rAAV Cdk2 promoter-less construct. (C) Graphic depiction of the rAAV-targeting strategy to introduce siSN silent point-mutations into exon 2 and a point mutation at Thr-160 to Ala (T160A) of human Cdk2. Highlighted are the two crucial steps of ‘targeting’ (Cas9 + gRNA + ssDNA) and allele ‘restoration’ (+Cre). Note locations of gRNA, siSN and T160A. (D) Introduction of siSN sequence plus a gene mutation into one allele of a given gene allows for selective depletion of the wild type allele by siWT resulting in a mutant phenotype, selective depletion of the mutant gene by siSN resulting in a rescued phenotype or depletion of both alleles to mimic a loss of function phenotype.
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).

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