A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells

• Published in: PloS one Vol. 11; no. 4; p. e0153901
• Main Authors: Findlay, Scott D, Vincent, Krista M, Berman, Jennifer R, Postovit, Lynne-Marie
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18.04.2016; Public Library of Science (PLoS)
• Subjects: Antibiotics; Assaying; Biology and Life Sciences; Cancer; Cloning; CRISPR; CRISPR-Cas Systems - genetics; Dentistry; Deoxyribonucleic acid; DNA; DNA Mutational Analysis - methods; Engineering and technology; Frizzled-related protein 1; Gene expression; Genetic Engineering; Genetic Loci; Genetic modification; Genome editing; Genomes; Genomics; High-Throughput Screening Assays - methods; Humans; Medical screening; Medicine; Molecular biology; Mutation; Mutation - genetics; Nuclease; Oncology; Physiological aspects; Plasmids; Polymerase chain reaction; Polymerase Chain Reaction - methods; Proteins; Research and Analysis Methods; RNA Editing; Single-nucleotide polymorphism; Stem cells; Transcription activator-like effector nucleases; Canada; United States; Edmonton Alberta Canada; Ontario Canada
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0153901

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.