Аннтотация

Despite rapid progress, many problems and limitations persist and limit 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- 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 as “ENIT” for Engineered Nucleases-Induced Translocations, 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, 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.

(PDF) A one-step PCR-based assay to evaluate efficiency and precision of genomic DNA-editing tools. Available from: https://www.researchgate.net/publication/314714362_A_one-step_PCR-based_assay_to_evaluate_efficiency_and_precision_of_genomic_DNA-editing_tools [accessed Dec 25 2018].