A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic D...
Science Direct June 16 2017
Diego Germini, Yara Bou Saada, Tatiana Tsfasman, Kristina Osina, Chloé Robin, Nikolay Lomov, Mikhail Rubtsov, Nikolajs Sjakste, Mars Lipinski, and Yegor Vassetzky
Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-editing tool to be tested is co-transfected into carefully chosen target cells along with that for an engineered nuclease of known specificity and efficiency. If the new enzyme efficiently cuts within the desired region, then specific chromosomal translocations will be generated between the two targeted genomic regions and be readily detectable by a one-step PCR or qPCR assay. The PCR product thus obtained can be directly sequenced, thereby determining the exact position of the double-strand breaks induced by the gene-editing tools. As a proof of concept, ENIT was successfully tested in different cell types and with different meganucleases, TALENs, and CRISPR/Cas9-based editing tools.
The field of in vivo gene editing using engineered nucleases is in strong development with the constantly increasing panel of available tools and techniques for testing their efficiency. Those gene-editing tools include meganucleases, transcription activator-like (TAL) effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system2.
Whatever the tools used, editing the genome is based on the generation of double-strand breaks (DSBs), which will trigger DNA repair, resulting in genome modifications either through homologous recombination (HR) or error-prone non-homologous end joining (NHEJ) processes. The modifications thus obtained include gene disruptions, insertions, substitutions, and chromosomal rearrangements.
The first and most important step in gene editing requires testing the efficiency and precision of the engineered nuclease. This is mostly based on detection of mismatches introduced in the target region by NHEJ. Various methods are employed to this aim, but they are all time-consuming and technically challenging.
Empire Genomic's IGH FISH Probe was used in this publication.
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