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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

CRISPR workflow.(a) Use of type IIS restriction enzymatic digestion enables rapid cloning of a custom CRISPR gRNA in a plasmid containing Cas9, mCherry and Neo resistance cassettes. Transformants of recombinant plasmids will be white, while transformants of non-recombinant plasmids will be blue. (b) Target cell lines are transfected with the Cas9+gRNA expression plasmid, enriched by cell sorting or antibiotic selection and expanded as single cell clones. (c) Schematic of droplet digital PCR-based screening of successful NHEJ.
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pone.0153901.g001: CRISPR workflow.(a) Use of type IIS restriction enzymatic digestion enables rapid cloning of a custom CRISPR gRNA in a plasmid containing Cas9, mCherry and Neo resistance cassettes. Transformants of recombinant plasmids will be white, while transformants of non-recombinant plasmids will be blue. (b) Target cell lines are transfected with the Cas9+gRNA expression plasmid, enriched by cell sorting or antibiotic selection and expanded as single cell clones. (c) Schematic of droplet digital PCR-based screening of successful NHEJ.

Mentions: For CRISPR/Cas-based editing, we first adapted an all-in-one CRISPR/Cas plasmid for rapid cloning using the type IIS restriction enzyme Esp3I and traditional blue/ white screening by inserting a LacZ-α open reading frame between two newly generated Esp3I restriction sites (Fig 1A). This plasmid allows for rapid single step cloning of any desired gRNA. It is a single plasmid system containing a mammalian antibiotic resistance gene and mCherry fluorescent protein for enrichment of transfected cells, if desired (Fig 1B). For TALEN-based editing, we used cloning methods previously described (see methods).


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)

CRISPR workflow.(a) Use of type IIS restriction enzymatic digestion enables rapid cloning of a custom CRISPR gRNA in a plasmid containing Cas9, mCherry and Neo resistance cassettes. Transformants of recombinant plasmids will be white, while transformants of non-recombinant plasmids will be blue. (b) Target cell lines are transfected with the Cas9+gRNA expression plasmid, enriched by cell sorting or antibiotic selection and expanded as single cell clones. (c) Schematic of droplet digital PCR-based screening of successful NHEJ.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153901.g001: CRISPR workflow.(a) Use of type IIS restriction enzymatic digestion enables rapid cloning of a custom CRISPR gRNA in a plasmid containing Cas9, mCherry and Neo resistance cassettes. Transformants of recombinant plasmids will be white, while transformants of non-recombinant plasmids will be blue. (b) Target cell lines are transfected with the Cas9+gRNA expression plasmid, enriched by cell sorting or antibiotic selection and expanded as single cell clones. (c) Schematic of droplet digital PCR-based screening of successful NHEJ.
Mentions: For CRISPR/Cas-based editing, we first adapted an all-in-one CRISPR/Cas plasmid for rapid cloning using the type IIS restriction enzyme Esp3I and traditional blue/ white screening by inserting a LacZ-α open reading frame between two newly generated Esp3I restriction sites (Fig 1A). This plasmid allows for rapid single step cloning of any desired gRNA. It is a single plasmid system containing a mammalian antibiotic resistance gene and mCherry fluorescent protein for enrichment of transfected cells, if desired (Fig 1B). For TALEN-based editing, we used cloning methods previously described (see methods).

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