Limits...
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.

Show MeSH

Related in: MedlinePlus

Genomic copy number alterations can be detected in ddPCR NHEJ detection assay.(a) Conceptual diagram displaying the expected changes in reference signal strength dependent on large deletions encompassing the reference probe binding site. (b) SFRP1 reference signal strength (copies/uL) in three C8161 SFRP1-edited cellular clones. (c) Copy number analysis of SFRP1 in three SFRP1-edited cellular clones normalized against RPP30. Error bars represent standard deviation (n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835065&req=5

pone.0153901.g004: Genomic copy number alterations can be detected in ddPCR NHEJ detection assay.(a) Conceptual diagram displaying the expected changes in reference signal strength dependent on large deletions encompassing the reference probe binding site. (b) SFRP1 reference signal strength (copies/uL) in three C8161 SFRP1-edited cellular clones. (c) Copy number analysis of SFRP1 in three SFRP1-edited cellular clones normalized against RPP30. Error bars represent standard deviation (n = 3).

Mentions: The quantitative power of the ddPCR assays led us to test their potential utility in other ways. First, we were interested if they were also capable of identifying large deletions that are occasionally obtained in genome editing experiments. If a deletion extends into any of the reference probe, forward primer, or reverse primer binding sites, these alleles should yield droplets that are also negative for the reference probe in addition to the NHEJ/drop-off probe. Indeed, when we tested gDNA from single cell-derived clones with either one or both SFRP1 alleles containing a large deletion, the number of total SFRP1 copies detected was 50% of the wild type for the single large deletion, and 0% (no copies detected) of the wild type for the sample with two large deletion-containing alleles (Fig 4).


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)

Genomic copy number alterations can be detected in ddPCR NHEJ detection assay.(a) Conceptual diagram displaying the expected changes in reference signal strength dependent on large deletions encompassing the reference probe binding site. (b) SFRP1 reference signal strength (copies/uL) in three C8161 SFRP1-edited cellular clones. (c) Copy number analysis of SFRP1 in three SFRP1-edited cellular clones normalized against RPP30. Error bars represent standard deviation (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153901.g004: Genomic copy number alterations can be detected in ddPCR NHEJ detection assay.(a) Conceptual diagram displaying the expected changes in reference signal strength dependent on large deletions encompassing the reference probe binding site. (b) SFRP1 reference signal strength (copies/uL) in three C8161 SFRP1-edited cellular clones. (c) Copy number analysis of SFRP1 in three SFRP1-edited cellular clones normalized against RPP30. Error bars represent standard deviation (n = 3).
Mentions: The quantitative power of the ddPCR assays led us to test their potential utility in other ways. First, we were interested if they were also capable of identifying large deletions that are occasionally obtained in genome editing experiments. If a deletion extends into any of the reference probe, forward primer, or reverse primer binding sites, these alleles should yield droplets that are also negative for the reference probe in addition to the NHEJ/drop-off probe. Indeed, when we tested gDNA from single cell-derived clones with either one or both SFRP1 alleles containing a large deletion, the number of total SFRP1 copies detected was 50% of the wild type for the single large deletion, and 0% (no copies detected) of the wild type for the sample with two large deletion-containing alleles (Fig 4).

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