A new paper published in Cell Reports describes how Single Molecule, Real-Time (SMRT®) Sequencing can be used to greatly improve outcome reporting for a variety of popular genome-editing approaches.
“Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing” comes from lead authors Ayal Hendel and Eric Kildebeck from the Porteus lab at Stanford University, along with other collaborators at Stanford and the Georgia Institute of Technology. The goal for this study was to contribute to the tremendous innovations occurring in the genome editing field — from CRISPR to TALENs and more — by finding a better tool to measure results of the editing procedures.
“A variety of reporter assays for tracking genome editing outcomes have been developed, but previously none have allowed for the frequency of different genome editing outcomes to be measured simultaneously at any endogenous locus of the investigator’s choosing,” the authors report. They turned to SMRT Sequencing and developed “a method for tracking genome editing outcomes at any site of interest.”
The challenge with measuring the results of genome editing, according to the paper, is that when a new set of reagents is developed, “the activity levels of nucleases and the frequency of the desired gene editing event must be determined and often need to be optimized for the specific cell type and system used by the researcher.” Current reporting tools include gel-based assays, fluorescent reporters, clone analysis, and more. “While each of these assays can provide a piece of the puzzle, they are often limited by the inability to measure the desired gene editing outcome directly, the need for reporter cell lines to optimize gene editing conditions, and limitations in detection sensitivity for difficult applications,” the authors note.
With its ultra-long reads, SMRT Sequencing performed well in tests that directly measured the results of genome-editing experiments. The scientists used a particularly active pair of TALENs (Transcription Activator-Like Effector Nucleases) to generate site-specific double-stranded breaks and introduce several point mutations. Then, they used SMRT Sequencing on the region of interest to measure non-homologous end-joining (NHEJ) and homology-directed repair (HDR) events. The method was found to be highly reproducible and showed excellent concordance with orthogonal validation methods.
The scientists then demonstrated the broad applicability of the SMRT Sequencing-based approach by applying it to more difficult experimental platforms such as human primary cells. They also measured the activities of different classes of nucleases at multiple genomic sites, optimized for different parameters of gene editing efficiencies, and demonstrated the detection of rare mutations including large insertions and deletions hundreds of base pairs in length. Indeed, the long-read sequencing proved to be particularly useful for measuring effectiveness of long donor DNA templates, which increase the efficiency of gene editing.
“SMRT DNA sequencing provides a rapid, quantitative, and sensitive strategy for tracking genome editing outcomes at endogenous loci,” the scientists conclude. “With the flexibility to evaluate new engineered nucleases and targeting constructs directly at desired loci without the development of reporter systems, SMRT DNA sequencing can help researchers minimize the time from conception to realization of their genome editing goal and drive this field even faster.”