A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools

Abstract

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.

HIV-1 Tat protein induces DNA damage in human peripheral blood B-lymphocytes via mitochondrial ROS production

“…They were treated with Tat or Tat together with Tempol at the indicated concentrations or left untreated for 5 days. Chromosome spreads were prepared as previously described [68] and analyzed at the Hôpital Saint Antoine-Paris, France.…”

Direct ENIT: An easy and reliable tool for gRNA efficacy verification by tracking induced chromosomal translocation

“…This modification results in a less time- and resource-consuming approach because it does not require genomic DNA isolation and purification steps and requires fewer cells per reaction. The direct, nested PCR strategy improves the sensitivity of the ENIT approach, allowing the detection of as few as 100 cells with induced translocation within a general population of 10,000 cells (compared with 700 cells with translocation among a general population of 7000 cells, in the original protocol [1] ). The attained sensitivity was comparable to those of other assays used for gRNA efficacy testing, including T7-assay (0.5%) and SURVEYOR (3%) [6] .…”

“…The engineered nuclease-induced translocations (ENIT) approach represents one of the easiest existing methods for confirming gRNA efficacy [1] . This method relies on the detection of nuclease-induced chromosomal translocations.…”

A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells

“…In addition, many other alternatives have been developed with improvement in certain aspects, including qEva-CRISPR 21 , engineered nuclease-induced translocations(ENIT) 22 , Cas9 nuclease based restriction fragment length polymorphism (RFLP) analysis 23 , Indel Detection by Amplicon Analysis (IDAA) 24 and the gene-editing frequency digital PCR (GEF-dPCR) 25 . However, most methods are multistep and quantify the editing efficiency based on pre-amplified PCR product coming from genomic DNA but not directly on the genomic DNA itself 9–20,22–24 . Sequence and length-dependent bias introduced during PCR amplification will unavoidably affect the detection accuracy 26–28 .…”