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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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Application of ddPCR NHEJ detection assay to determine genome editing efficiencies of NH versus NN RVD-containing TALENs.(a) TALENs were designed against an SFRP1 target; NH and NN RVDs were used as illustrated. (b,c) Mutant alleles were detected by the ddPCR NHEJ detection assay in bulk population gDNA of C8161 SFRP1-edited cells. Technical (b) and biological (c) replicates illustrate the enhanced gene editing ability of the NN RVD-containing TALEN pair to this target. Points represent percent mutant alleles detected; error bars represent 95% confidence intervals.
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pone.0153901.g005: Application of ddPCR NHEJ detection assay to determine genome editing efficiencies of NH versus NN RVD-containing TALENs.(a) TALENs were designed against an SFRP1 target; NH and NN RVDs were used as illustrated. (b,c) Mutant alleles were detected by the ddPCR NHEJ detection assay in bulk population gDNA of C8161 SFRP1-edited cells. Technical (b) and biological (c) replicates illustrate the enhanced gene editing ability of the NN RVD-containing TALEN pair to this target. Points represent percent mutant alleles detected; error bars represent 95% confidence intervals.

Mentions: Second, we demonstrated that these assays can be used as a quantitative platform to directly test the performance of distinct genome editing systems. We chose to compare the efficiency of TALEN-induced mutations using either NN or NH repeat variable diresidues (RVDs) to bind G nucleotides at the SFRP1 locus. NN was initially the preferred RVD for the targeting of guanine (G) nucleotides since it was recognized as a “strong repeat” important for the efficacy of TALEN constructs [18]. However, NN was also known to lack desired single nucleotide specificity as it also efficiently recognized adenine (A) nucleotides [19]. More recently, the NH RVD was adopted as an NN substitute to achieve increased specificity for guanine targeting [18,20]. Using the first coding exon of SFRP1 as an example target locus, our ddPCR assay was used to demonstrate that the NN-containing TALEN pair was much more efficient at inducing mutations than its NH-containing counterpart across three independent experiments (P = 0.0084 by t-test) (Fig 5). An average of 26% of alleles were mutated using the NN-containing TALEN pair, compared to an average of 2% of alleles when the NH-containing TALEN pair was employed. We demonstrate that the technical variability in this assay is minimal, and has the benefit of a statistical confidence interval associated with each individual well, making it possible to quantify potentially small differences in mutation frequency between samples.


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)

Application of ddPCR NHEJ detection assay to determine genome editing efficiencies of NH versus NN RVD-containing TALENs.(a) TALENs were designed against an SFRP1 target; NH and NN RVDs were used as illustrated. (b,c) Mutant alleles were detected by the ddPCR NHEJ detection assay in bulk population gDNA of C8161 SFRP1-edited cells. Technical (b) and biological (c) replicates illustrate the enhanced gene editing ability of the NN RVD-containing TALEN pair to this target. Points represent percent mutant alleles detected; error bars represent 95% confidence intervals.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153901.g005: Application of ddPCR NHEJ detection assay to determine genome editing efficiencies of NH versus NN RVD-containing TALENs.(a) TALENs were designed against an SFRP1 target; NH and NN RVDs were used as illustrated. (b,c) Mutant alleles were detected by the ddPCR NHEJ detection assay in bulk population gDNA of C8161 SFRP1-edited cells. Technical (b) and biological (c) replicates illustrate the enhanced gene editing ability of the NN RVD-containing TALEN pair to this target. Points represent percent mutant alleles detected; error bars represent 95% confidence intervals.
Mentions: Second, we demonstrated that these assays can be used as a quantitative platform to directly test the performance of distinct genome editing systems. We chose to compare the efficiency of TALEN-induced mutations using either NN or NH repeat variable diresidues (RVDs) to bind G nucleotides at the SFRP1 locus. NN was initially the preferred RVD for the targeting of guanine (G) nucleotides since it was recognized as a “strong repeat” important for the efficacy of TALEN constructs [18]. However, NN was also known to lack desired single nucleotide specificity as it also efficiently recognized adenine (A) nucleotides [19]. More recently, the NH RVD was adopted as an NN substitute to achieve increased specificity for guanine targeting [18,20]. Using the first coding exon of SFRP1 as an example target locus, our ddPCR assay was used to demonstrate that the NN-containing TALEN pair was much more efficient at inducing mutations than its NH-containing counterpart across three independent experiments (P = 0.0084 by t-test) (Fig 5). An average of 26% of alleles were mutated using the NN-containing TALEN pair, compared to an average of 2% of alleles when the NH-containing TALEN pair was employed. We demonstrate that the technical variability in this assay is minimal, and has the benefit of a statistical confidence interval associated with each individual well, making it possible to quantify potentially small differences in mutation frequency between samples.

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