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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

Droplet digital PCR can be used to screen for indels in bulk-edited populations and single cell clones.(a) Example ddPCR plots of parental population gDNA and bulk-edited population gDNA for three given genomic targets. (b) Conceptual and actual results of a T7E1 digestion resolved by gel electrophoresis for wildtype, mono-allelic mutant and bi-allelic mutant clones. (c) ddPCR plots of wildtype, mono-allelic mutant and bi-allelic mutant clones. Note that FAM and HEX are used for different probes between the SFRP1 and NODAL assays.
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pone.0153901.g002: Droplet digital PCR can be used to screen for indels in bulk-edited populations and single cell clones.(a) Example ddPCR plots of parental population gDNA and bulk-edited population gDNA for three given genomic targets. (b) Conceptual and actual results of a T7E1 digestion resolved by gel electrophoresis for wildtype, mono-allelic mutant and bi-allelic mutant clones. (c) ddPCR plots of wildtype, mono-allelic mutant and bi-allelic mutant clones. Note that FAM and HEX are used for different probes between the SFRP1 and NODAL assays.

Mentions: We demonstrate the ability of ddPCR-based assays to successfully detect nuclease-induced mutations in our sample targets. These assays consist of a duplexed primer probe based ddPCR assay in which one probe binds a “reference” sequence distant from the nuclease target site but still within the amplicon, and a second “NHEJ/drop-off” probe binds at the nuclease target site. In a 2-dimensional view of the ddPCR analysis, droplets containing signal from both probes contain wild-type amplicons, while droplets containing signal from the reference probe but not the NHEJ/drop-off probe contain amplicons with mutations at the target site (Fig 1C). We very rarely detected droplets that could be classified as containing target mutations in control-transfected or parental samples, and successfully detected nuclease-induced mutations in CRISPR or TALEN-treated cells (Fig 2A). For single cell-derived clones, the same assays reliably detected induced mutations. Unlike mismatch nuclease assays, our ddPCR assays were definitively able to distinguish samples with mono-allelic versus bi-allelic mutations (Fig 2B and 2C). All ddPCR assays were validated by sequencing single cell-derived clones (see methods). No mutant sequences were detected in samples with virtually all droplets clustering as double positive/ wild type. Both mutant sequences and wild type sequences were detected in samples with both wild type and NHEJ droplets. No wild type sequences were detected in samples with virtually all droplets clustering as NHEJ droplets (Fig 2C).


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)

Droplet digital PCR can be used to screen for indels in bulk-edited populations and single cell clones.(a) Example ddPCR plots of parental population gDNA and bulk-edited population gDNA for three given genomic targets. (b) Conceptual and actual results of a T7E1 digestion resolved by gel electrophoresis for wildtype, mono-allelic mutant and bi-allelic mutant clones. (c) ddPCR plots of wildtype, mono-allelic mutant and bi-allelic mutant clones. Note that FAM and HEX are used for different probes between the SFRP1 and NODAL assays.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153901.g002: Droplet digital PCR can be used to screen for indels in bulk-edited populations and single cell clones.(a) Example ddPCR plots of parental population gDNA and bulk-edited population gDNA for three given genomic targets. (b) Conceptual and actual results of a T7E1 digestion resolved by gel electrophoresis for wildtype, mono-allelic mutant and bi-allelic mutant clones. (c) ddPCR plots of wildtype, mono-allelic mutant and bi-allelic mutant clones. Note that FAM and HEX are used for different probes between the SFRP1 and NODAL assays.
Mentions: We demonstrate the ability of ddPCR-based assays to successfully detect nuclease-induced mutations in our sample targets. These assays consist of a duplexed primer probe based ddPCR assay in which one probe binds a “reference” sequence distant from the nuclease target site but still within the amplicon, and a second “NHEJ/drop-off” probe binds at the nuclease target site. In a 2-dimensional view of the ddPCR analysis, droplets containing signal from both probes contain wild-type amplicons, while droplets containing signal from the reference probe but not the NHEJ/drop-off probe contain amplicons with mutations at the target site (Fig 1C). We very rarely detected droplets that could be classified as containing target mutations in control-transfected or parental samples, and successfully detected nuclease-induced mutations in CRISPR or TALEN-treated cells (Fig 2A). For single cell-derived clones, the same assays reliably detected induced mutations. Unlike mismatch nuclease assays, our ddPCR assays were definitively able to distinguish samples with mono-allelic versus bi-allelic mutations (Fig 2B and 2C). All ddPCR assays were validated by sequencing single cell-derived clones (see methods). No mutant sequences were detected in samples with virtually all droplets clustering as double positive/ wild type. Both mutant sequences and wild type sequences were detected in samples with both wild type and NHEJ droplets. No wild type sequences were detected in samples with virtually all droplets clustering as NHEJ droplets (Fig 2C).

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