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

Abstract

CRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3′ end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.

A dinucleotide tag-based parallel reporter gene assay method

“…We previously reported that using primers with the tag-specific nucleotide placed at its 3’ end could achieve sequence-specific PCR quantification, and the nucleotide type of tag could significantly affect the PCR specificity [25]. To determine the best dinucleotide barcoding rule, we constructed 16 plasmids containing all the 16 kinds of dinucleotides using the coding sequence of the HOXB13 gene ( Fig.…”

A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution

“…3b and 3e). The potential mechanism that enables DST-PCR to detect these types of mutations is that the mismatched nucleotide at the primer 3' end was removed by the proofreading 3' to 5' exonuclease activity of the high-fidelity DNA polymerases [26][27][28] such as ExTaq DNA polymerase and KOD-Plus-Neo DNA polymerase during polymerizing and hence restored the PCR amplification capacity in part or completely. This means that DNA polymerases play an important role in deciding the discrimination ability of DST-PCR with high-fidelity DNA polymerase, increasing its ability in distinguishing the indels.…”

rs10514231 Leads to Breast Cancer Predisposition by Altering ATP6AP1L Gene Expression

“…We then performed CRISPR/Cas9-mediated genome editing on the higher activity C allele in heterozygous MDA-MB-453 cells to test the direct regulatory effect of rs10514231 on gene expression. The genome-editing efficiency on the C allele was around 60%, as determined by the getPCR method [16] (Figure 3d). We analyzed the expression level of the three potential eQTL genes through RT-qPCR and found that only ATP6AP1L expression was affected by the genome-editing event and had a 40% reduction in the edited cells (Figure 3e-g).…”

“…The medium was changed with the fresh medium containing 2 µg/mL puromycin 24 h later. After the non-transfected cells died completely, the survived transfected cells were passaged, with a part used for the editing efficiency evaluation using the getPCR method, as described previously [16]. Single-cell clones were isolated using the limited dilution method in 96-well plates, and the genotypes were determined by the getPCR assay and confirmed through Sanger sequencing.…”