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.

Keywords: CRISPR/Cas9; PCR; TALEN; assay; translocations.

Graphical abstract

Figure 1

PCR Detection of a Rearranged DNA Sequence Resulting from t(8;14) Chromosomal Translocation (A) PCR on DNA extracted from untransfected HeLa cells. The represented amplicons were obtained using the following primer pairs: lane 1, GAPDH F + R; lane 2, MYC F + R; lane 3, IGH F + R; lane 4, MYC F + IGH R. M is the molecular weight marker. (B) PCR on DNA extracted from transfected HeLa cells with the four TALENS recognizing the MYC and IGH genes. The represented amplicons were obtained using the following primer pairs: lane 1, GAPDH F + R; lane 2, MYC F + R; lane 3, IGH F + R; lane 4, MYC F + IGH R. The experiments were repeated three times and representative gels are displayed. (C and D) Alignment of IGH (C) or MYC (D) wild-type NCBI reference sequences (NG_001019.5 and NG_007161.1, respectively) with the sequence of the amplicon obtained from DNA extracted from transfected HeLa cells and amplified with MYC F + IGH R primers. The part of the alignment where the sequences are perfectly aligned is highlighted in light gray, whereas the parts of the sequences containing mismatches (in bold) next to the TALENs cutting site and next to the place where the translocation occurred are highlighted in dark gray.

Figure 2

Chromosome Spread of HeLa Cells Representative FISH image of a chromosome spread of one HeLa cell transfected with the four TALENS targeting the MYC and IGH genes. The chromosome indicated by the red arrow exhibits the t(8:14) translocation, as evidenced by colocalized MYC (red) and IGH (green) signals. Left: enlarged image of the metaphase chromosome containing the t(8:14) translocation. At least 100 metaphases were analyzed to detect one translocation.

Figure 3

Sensitivity of the PCR-Based Detection of Translocations (A) Intensity of bands obtained by the PCR amplification of serial dilutions of the DNA obtained from HeLa cells transfected with the four TALENS recognizing the MYC and IGH regions. The amplicons were obtained with MYC F and IGH R primers. (B) The intensity of each band was related to the number of transfected cells from which we obtained the DNA used for PCR. The reliability of dilutions was calculated by amplifying the same samples with MYC F + R and applying the same method of quantification. Error bars represent the variation of band intensities obtained in one transfection experiment. The experiment was repeated twice, and representative graphs from one experiment are displayed.

Figure 4

ENIT Can Be Used to Detect the Efficiency of Various Gene-Editing Tools and Targets (A) PCR on DNA extracted from HeLa cells transfected with the four TALENS recognizing the MYC and 4q (FSHD) regions. The represented amplicons were obtained using the following primer pairs: lane 1, 4q F+R; lane 2, MYC F + R; lane 3, MYC F + 4q R. (B) PCR on DNA extracted from untransfected HeLa cells. The represented amplicons were obtained using the following primer pairs: lane 1, 4q F + R; lane 2, MYC F + R; lane 3, MYC F + 4q R. (C) PCR on DNA extracted from HeLa cells transfected with the two TALENS recognizing the MYC region and the CRISPR/Cas9 for the EZH2 gene region. The represented amplicons were obtained using the following primer pairs: lane 1, MYC F + R; lane 2, EZH2 F + R; lane 3, EZH2 F + MYC R. (D) PCR on DNA extracted from untransfected HeLa cells. The represented amplicons were obtained using the following primer pairs: lane 1, MYC F + R; lane 2, EZH2 F + R; lane 3, EZH2 F + MYC R.