Digital detection of endonuclease mediated gene disruption in the HIV provirus

Abstract

Genome editing by designer nucleases is a rapidly evolving technology utilized in a highly diverse set of research fields. Among all fields, the T7 endonuclease mismatch cleavage assay, or Surveyor assay, is the most commonly used tool to assess genomic editing by designer nucleases. This assay, while relatively easy to perform, provides only a semi-quantitative measure of mutation efficiency that lacks sensitivity and accuracy. We demonstrate a simple droplet digital PCR assay that quickly quantitates a range of indel mutations with detection as low as 0.02% mutant in a wild type background and precision (≤6%CV) and accuracy superior to either mismatch cleavage assay or clonal sequencing when compared to next-generation sequencing. The precision and simplicity of this assay will facilitate comparison of gene editing approaches and their optimization, accelerating progress in this rapidly-moving field.

Figure 1. Cleavage of HIV pol by an engineered megaTAL.

(A) Location of the megaTAL cleavage site (S20, red triangle) within the HIV provirus. (B) Tal effector (TALE) and meganuclesase domains for the HIV pol-specific 6.5 or 7.5 RVD repeat containing megaTALs alongside their HIV pol target sequences. TALE binding (red), S20 meganuclease binding (blue) and S20 meganuclease cleavage (underlined) sites are shown. NTD – N-terminal domain; CTD – C-terminal domain.

Figure 2. Droplet digital PCR can detect a wide range of deletion mutations.

(A) Primer and probe design. HIV pol-specific forward and reverse primers are used with a reference (1) and target (2) probe that bind to opposite strands of the same PCR amplicon. The target probe (2) is centered on the megaTAL target site, indicated by a red triangle. (B) Target site deletion mutations used for ddPCR assay validation. (C) Two dimensional ddPCR amplitude plot showing that the assay detects the reference sequence in addition to wild type target sequence (X), one base pair deletions (Y), or 2, 3, 4 and 7 base pair deletions (Z) at the megaTAL target site in reference plasmids. Mutant control plasmids were spiked into a background of wild type plasmid at an approximate ratio of 85:1:2 (X:Y:Z). Droplets containing no target are shown in gray. (D) Two dimensional ddPCR amplitude plot showing that the assay detects the reference sequence, wild type (X), and mutant (Y, Z) sequences in 293T cells 72 hours after transfection with pDHIV3 and plasmids expressing the 6.5 megaTAL and Trex2. (E) 293T cells with pDHIV3 and no megaTAL treatment (negative control) have a very low false mutation rate; >99% of reference positive droplets are also target positive (X).

Figure 3. Lower range of detection for a mutant plasmid (2 base pair deletion)

in a background of wild type plasmid.Each point represents the mean of 3 reactions. All reactions were non-negative for mutant down to 0.02% mutant. Error bars indicate one standard deviation.

Figure 4. Scatter plot of false mutation rate detected from reference cell line genomic DNA (SupT1-HIV-WT sample).

The red dashed line indicates the empirically determined Limit of Blank (LoB).

Figure 5. Mutation detection assay comparison

(A) T7 endonuclease I MCA mutation detection. S20 target site-specific PCR amplicon size and predicted cleavage products.(B) 293T cells were transfected with pDHIV3 and plasmids expressing the indicated megaTALs +/− a Trex2 expressing plasmid. At 72 hours post transfection DNA was extracted and PCR amplicons spanning the target site were amplified for analysis of target site-disruption. (C) Comparison of the T7 mismatch cleavage assay (T7 MCA), clone sequencing (clone), illumina sequencing (NGS), and droplet digital PCR (ddPCR) mutation detection in megaTAL treated 293T cells transfected with pDHIV3 +/− Trex2 treatment. Error bars for ddPCR represent the standard deviation of three replicate samples.