Category: Assay Development and Screening

1163-D - Fast and reliable method for detecting base editing in clonal cell lines

Tuesday, February 6, 2018
5:00 PM - 6:00 PM

One of the most powerful applications of genome editing is the introduction of base changes in specific genomic sites to mimic single-nucleotide polymorphisms (SNPs) related to human diseases or introducing stop codons to generate precise gene knockouts. However, screening large number of clones to identify edited clonal cell lines containing the engineered base of interest is still a bottleneck, especially if no phenotypic readout is applicable. Sanger sequencing is a potential approach to detect SNPs, but it is not easy to apply in a high-throughput manner; next generation sequencing, in contrast, allows researchers to screen 96-well plates but at a far higher cost. To address this need, we developed a simple SNP-detection method that allows for rapid screening of clones from 96-well plates. Our assay comprises PCR amplification of the target site, followed by an enzymatic assay and a fluorescence based read out using a standard plate reader. No additional special instrumentation is required. The overall workflow takes approximately four hours and any positive fluorescent signal is highly correlated with the successful introduction of the desired SNP. This method allows for the detection of edited clonal cell lines independent of the engineered nucleotide substitution and the surrounding targeted genomic loci. As a proof of concept, we have applied this method to successfully detect all the possible transitions in several human loci using genomic DNA as template. As a final test, several nucleotide exchanges have been detected directly in cultured human fibroblasts.

Liz Quinn

Associate Director for Stem Cell Marketing
Takara Bio USA, Inc.
Mountain View, CA

Dr. Liz Quinn is the Associate Director for Stem Cell Marketing at Takara Bio USA following nearly 20 years of research and commercial experience in the life science and biotechnology industries. Dr. Quinn joined Clontech Laboratories, Inc. in 2001 and held varied responsibility within Operations, R&D and Marketing. In 2008, she joined DiscoverX Corporation and managed the GPCR drug discovery business. After leaving DiscoverX in 2014, she served as founder and CEO for a new life science and drug discovery-focused marketing consulting company based in Northern CA. Then in 2015, Liz joined Takara Bio USA where she has focused on launching a new stem cell product portfolio which includes human iPS and ES-derived lines and culture systems for gene editing. In addition to her current role, Liz is a board member of ELRIG, the European Laboratory Research and Innovation Group, and has served as their Scientific Programme Director since 2014.