title: A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells.
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.
authors.affiliation: Department of Anatomy and Cell Biology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.|Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Findlay
authors.affiliation: Department of Anatomy and Cell Biology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.|Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Krista M
authors.initials: KM
authors.lastname: Vincent
authors.affiliation: Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Berman
authors.affiliation: Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Lynne-Marie
authors.initials: LM
authors.lastname: Postovit
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005818:Genetic Engineering; D056426:Genetic Loci; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017393:RNA Editing