Detection and Quantification of HDR and NHEJ Induced by Genome Editing at Endogenous Gene Loci Using Droplet Digital PCR

Abstract

Genome editing holds great promise for experimental biology and potential clinical use. To successfully utilize genome editing, it is critical to sensitively detect and quantify its outcomes: homology-directed repair (HDR) and nonhomologous end joining (NHEJ). This has been difficult at endogenous gene loci and instead is frequently done using artificial reporter systems. Here, we describe a droplet digital PCR (ddPCR)-based method to simultaneously measure HDR and NHEJ at endogenous gene loci. This highly sensitive and quantitative method may significantly contribute to a better understanding of DNA repair mechanisms underlying genome editing and to the improvement of genome editing technology by allowing for efficient and systematic testing of many genome editing conditions in parallel.

Keywords: CRISPR/Cas9; Genome editing; HDR; NHEJ; TALEN; ddPCR.

Fig. 1

Separated cut and mutation sites: Assay design, example target sequence, and 2D droplet plot of edited genomic DNA. Depending on the editing strategy and the relative positions of cut site and edit site, the positions of assay probes and the need for a competitive blocking (dark) probe vary. The reference and HDR probes are constant regardless of the location or number of cut sites. (a) Assay design when cut site and mutation site are separated. Outcomes for three different alleles are shown (retention of WT sequence, alteration of a single base via HDR, or indel via NHEJ). The HDR and NHEJ probes are located on nonoverlapping mutation and cut sites, respectively. To prevent nonspecific binding of the HDR probe to the original “WT” sequence, a nonfluorescent (dark) “WT” probe is included that competes with the HDR probe for WT allele binding (see Figs. 2, 4, and 5 for other cases). (b) Assay design when cut site and mutation site overlap. See Fig. 4 legend for details. (c) Assay design when two cut sites are introduced. See Fig. 5 legend for details

Fig. 2

Comparison of three ddPCR assay designs for three different editing designs. (a) Assay design when cut site and mutation site are separated. See Fig. 1a legend for details. (b) Assay design when two cut sites are introduced. See Fig. 4 legend for details. (c) Assay design when cut site and mutation site overlap. See Fig. 5 legend for detail

Fig. 3

Separated cut and mutation sites, with one NHEJ probe: Assay design and 2D droplet plot with droplet group definitions for analysis. Details as in Fig. 1a, c

Fig. 4

Cut site and mutation site overlap. (a) Assay design when cut site and mutation site overlap. In these cases, the NHEJ probe overlaps with and is on the same strand as the HDR probe. The NHEJ probe thus competes with the HDR probe for WT binding and a dark probe is not necessary. Also, the HDR allele becomes FAM++ and HEX−. (b) Other droplet populations defined as in Fig. 1c

Fig. 5

Two cut sites are introduced: ddPCR assay design and 2D droplet plot. (a) Assay design when two cut sites are introduced. Because dual Cas9 systems introduce two cuts, two NHEJ probes are included in the assay to detect possible indels on either side of the mutation site. When neither NHEJ probe competes with the HDR probe—as shown here—a dark probe is also designed to avoid binding of the HDR probe to the WT allele. Because two NHEJ probes are included in this assay, the WT allele is detected as FAM+ and HEX++, and the NHEJ alleles are detected as FAM+ and HEX+, or FAM+ and HEX−. (b) Two-dimensional plot of an assay with two NHEJ probes. Definitions of N_Empty and N_HDR+ populations are the same as in Fig. 1c. However, because two NHEJ probes are included in this assay, there are two droplet populations containing only NHEJ alleles—one that lost one of the two NHEJ probe binding sites of NHEJ probes (FAM+ HEX+) and one that lost both (FAM+ HEX−). All other droplets were gated as N_WT+ population (FAM+ HEX++). These definitions are used to calculate the HDR and NHEJ allelic frequencies (see Subheading 3.4)

Fig. 6

Different HDR and NHEJ frequencies induced by TALEN in RBM20 in HEK293 cells and human induced pluripotent stem cells. (a and b) HDR and NHEJ allelic frequencies induced by the same TALENs targeting RBM20 in HEK293 cells (a) and human induced pluripotent stem cells (b). The NHEJ and HDR probes directly compete to each other, so N_HDR+ population was FAM++ HEX− as in Fig. 4. The frequencies are shown in the two-dimensional plots. The two different cell types showed different HDR and NHEJ frequencies