However, with this strategy only clones having the large deletion in all alleles are discriminated, and the potentially useful mutant clones having only small indels are ignored, reducing the success rate of obtaining mutants. The usage of Cas9 nuclease with two guide RNAs targeting relatively distant sites enables an easy discrimination by inducing a bigger deletion detectable by PCR (not to be confounded with the double-nickase strategy where the offset should be small enough to get a double strand break ). The indels caused by CRISPR/Cas9 editing are often small, thus standard PCR with genomic DNA (gDNA) is not useful to detect the mutants since the amplicon sizes are not different enough. Therefore, a strategy to identify mutant clones quickly and accurately is very important.
For this reason, researchers have often performed isolation of mutant clones before doing their assay of interest. However, even with the most efficient delivery strategies, gene disruption does not reach 100% efficiency in cell culture.
This often leads to the introduction of indels, resulting in an efficient loss of function of gene. In the widely-used CRISPR/Cas9 system derived from Streptococcus pyogenes, the Cas9 nuclease creates a double strand break in the genome at a site complementary to the guide RNA, which is then repaired by various repair machineries of the cell. Recently, genome editing by the CRISPR/Cas9 system has became a widely used strategy to study gene functions in cells and in vivo. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. TH was supported by the Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowships for Research Abroad. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was supported by the Swiss National Science Foundation (SNSF) the National Centre of Competence in Research (NCCR) Chemical Biology SystemsX (evaluated by the SNSF) and the Canton of Geneva. Received: FebruAccepted: Published: June 6, 2017Ĭopyright: © 2017 Harayama, Riezman. PLoS ONE 12(6):Įditor: Hodaka Fujii, Osaka University, JAPAN The provided method is easy to perform and requires only basic laboratory equipment, making it suitable for almost all laboratories.Ĭitation: Harayama T, Riezman H (2017) Detection of genome-edited mutant clones by a simple competition-based PCR method. Finally, we applied this method to show that mutations conferring drug resistance can be detected with high accuracy.
Antibody repertoire microbiome how to#
Together, we show how to optimize primer design in order to improve the effectiveness of the discrimination. Here, we show that knockout cells can be discriminated by a competition-based PCR, using a mixture of three primers, among which one primer overlaps with the Cas9 cleavage site. Therefore, a method to discriminate accurately mutated clones easily and quickly is crucial to accelerate the research using CRISPR/Cas9. Since the efficiency of gene disruption in cell culture does not reach 100% typically, cloning of mutant cells is often performed to obtain fully mutated cells. Genome editing by the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) system is a revolutionary strategy to study gene functions.
0 kommentar(er)
