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A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells.

Findlay SD, Vincent KM, Berman JR, Postovit LM - PLoS ONE (2016)

Bottom Line: Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity.Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies.Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

ABSTRACT
The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs "mismatch nucleases" T7E1 or "Surveyor" that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an "all-in-one" CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.

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Related in: MedlinePlus

Sensitivity of indel detection in mismatch and ddPCR assays.(a,b) Genomic DNA samples from wild type and target-disrupted cells were mixed in indicated ratios and subjected to a T7E1 cleavage assay. Gel analysis was conducted on SFRP1 (a) and NODAL (b) targets. DNA sequences of the mutant cellular clone are displayed at the top of the panel. Underlines indicate TALEN spacer region in a, and gRNA target (including PAM sequence) in b. (c-e) Genomic DNA from target-disrupted samples were added to wild type samples at indicated amounts and subjected to ddPCR analysis. Error bars indicate 95% confidence interval. Yellow line depicts the mutant copies expected. “N.D.” indicates none detected.
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pone.0153901.g003: Sensitivity of indel detection in mismatch and ddPCR assays.(a,b) Genomic DNA samples from wild type and target-disrupted cells were mixed in indicated ratios and subjected to a T7E1 cleavage assay. Gel analysis was conducted on SFRP1 (a) and NODAL (b) targets. DNA sequences of the mutant cellular clone are displayed at the top of the panel. Underlines indicate TALEN spacer region in a, and gRNA target (including PAM sequence) in b. (c-e) Genomic DNA from target-disrupted samples were added to wild type samples at indicated amounts and subjected to ddPCR analysis. Error bars indicate 95% confidence interval. Yellow line depicts the mutant copies expected. “N.D.” indicates none detected.

Mentions: We tested the performance of these assays by spiking in genomic DNA (gDNA) from a single-cell derived clone with bi-allelic mutations into a high concentration of non-mutated wild type gDNA. This allowed us to create carefully controlled samples representing a small number of mutated cells in a larger background of non-mutated cells, while maintaining the natural complexity, concentration, and purity of a typical gDNA sample. These samples were then subjected to both our ddPCR assays and standard mismatch nuclease assays for performance evaluation (Fig 3).


A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells.

Findlay SD, Vincent KM, Berman JR, Postovit LM - PLoS ONE (2016)

Sensitivity of indel detection in mismatch and ddPCR assays.(a,b) Genomic DNA samples from wild type and target-disrupted cells were mixed in indicated ratios and subjected to a T7E1 cleavage assay. Gel analysis was conducted on SFRP1 (a) and NODAL (b) targets. DNA sequences of the mutant cellular clone are displayed at the top of the panel. Underlines indicate TALEN spacer region in a, and gRNA target (including PAM sequence) in b. (c-e) Genomic DNA from target-disrupted samples were added to wild type samples at indicated amounts and subjected to ddPCR analysis. Error bars indicate 95% confidence interval. Yellow line depicts the mutant copies expected. “N.D.” indicates none detected.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835065&req=5

pone.0153901.g003: Sensitivity of indel detection in mismatch and ddPCR assays.(a,b) Genomic DNA samples from wild type and target-disrupted cells were mixed in indicated ratios and subjected to a T7E1 cleavage assay. Gel analysis was conducted on SFRP1 (a) and NODAL (b) targets. DNA sequences of the mutant cellular clone are displayed at the top of the panel. Underlines indicate TALEN spacer region in a, and gRNA target (including PAM sequence) in b. (c-e) Genomic DNA from target-disrupted samples were added to wild type samples at indicated amounts and subjected to ddPCR analysis. Error bars indicate 95% confidence interval. Yellow line depicts the mutant copies expected. “N.D.” indicates none detected.
Mentions: We tested the performance of these assays by spiking in genomic DNA (gDNA) from a single-cell derived clone with bi-allelic mutations into a high concentration of non-mutated wild type gDNA. This allowed us to create carefully controlled samples representing a small number of mutated cells in a larger background of non-mutated cells, while maintaining the natural complexity, concentration, and purity of a typical gDNA sample. These samples were then subjected to both our ddPCR assays and standard mismatch nuclease assays for performance evaluation (Fig 3).

Bottom Line: Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity.Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies.Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

ABSTRACT
The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs "mismatch nucleases" T7E1 or "Surveyor" that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an "all-in-one" CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.

Show MeSH
Related in: MedlinePlus