The development and widespread adoption of new genome editing techniques in basic and clinical research have yielded an impressive number of success stories and hold great promise for the treatment of diseases with a genetic component, gene therapy and synthetic biology. In particular, the recent explosion in genome editing due to CRISPR-Cas and similar systems (1) that enable previously unobtainable precision, has triggered an avalanche of innovative strategies for research into epigenetics, genome structure and gene expression and are rapidly improving our understanding on many fronts.
Two common validations are required in many genome editing projects. Firstly, researchers must confirm that the desired event, such as a knock-in, the down-regulation of a particular gene or a specific sequence mutation has occurred. Secondly, researchers often want to examine for off-target genome modifications, which could have confounding effects on their results — for example in systems with multi-copy genes or pseudogenes. Since the efficiency of many types of genome editing techniques is low, only a sub-population of the treated cells are likely to be modified, meaning that identifying successfully engineered cells within a bulk population for clonal expansion is also a key step of any experiment. Whole genome amplification (WGA) and transcriptome amplification (WTA) technologies, which allow the analysis of the entire genome or RNA expression from a single cell, are an essential tool for quality control and confirmation experiments in genome editing workflows. Three publications that applied WGA technology to confirm gene editing caught my attention.
In the context of genome editing, there is the concept of safe harbors, or locations in the genome where transgenes can be introduced with predictable and stable results. Yang et al (2) identified rbROSA26 as the first such locus in the rabbit genome, and generated two knock-in lines with CRISPR-Cas9. In their workflow, embryos were microinjected with the CRISPR-Cas9 construct and then clonally expanded. Single cells were removed at the blastocyst stage and WGA was used to generate sufficient template from each single cell for PCR-based confirmation of the knock-in event, allowing the researchers to ensure that editing was successful. Confirming successful editing early in a workflow allowed the researchers to focus on the expansion or implantation of only successfully edited embryos, reducing the cost and effort associated with carrying non-edited animals through the entire procedure.
In addition to being useful to confirm successful editing, researchers also used WGA/WTA to screen for off-target effects. In the field of HIV research, Peterson et al (3) used a zinc finger nuclease system to edit the CCR5 locus, which encodes a chemokine receptor necessary for the initial infection of cells by the HIV virus in humans. In this experiment, they isolated and cultured CD34+ hematopoietic stem cells and disrupted the CCR5 locus, then infused the cells back into conditioned recipients and tracked the engraftment and expansion of these modified cells. The group used REPLI-g WGA technology, followed by deep amplicon sequencing to track the population of cells arising from the grafts in vivo over time, and this analysis of off-target effects provided data to confirm the specificity and precision of the process.
New gene editing technologies can also be applied to high-throughput discovery work. Wong et al. (4) developed a CRISPR-Cas9-based screening platform that uses large sets of guide RNAs used in tandem for the systematic analysis of combinatorial gene function. They used the REPLI-g Single Cell Kit and targeted allele sequencing to confirm the successful editing of single FACS-sorted OVCAR8-ADR-Cas cells.
In addition to DNA-based validation, gene expression-based validation strategies — where researchers evaluate expression of a knock-in, for example — are also commonly used, and combined DNA/RNA approaches are also possible.
Regardless of the scale of the experiment, the confirmation of successful gene editing, the expression of knock-ins or the analysis of potential off-target effects are all important validation measures to ensure subsequent experiments are trustworthy. REPLI-g Single Cell WGA and WTA Kits are widely used to analyze event-specific genome sequence or gene expression, and require only a single cell as input. These kits allow you to determine successful genome editing from a single cell, limiting the amount of clonal expansion needed, allowing analysis prior to implantation in some experiments, and shortening your overall workflow time.
The applications presented here are for research use only. Not for use in diagnostic procedures.
- 1. Ledford, H. (2016) CRISPR: gene editing is just the beginning. 156 Nature Vol 531 (Link)
- 2. Yang, D. et al. (2016) Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression. Sci Rep. 6, 25161 (Link)
- 3. Peterson, C. et al. (2016) Long-term multilineage engraftment of autologous genome-edited hematopoietic stem cells in nonhuman primates. Blood. 127, 2416-2426 (Link)
- 4. Wong, A.S.L. et al. (2016) Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM. Proc Natl Acad Sci U S A. 113, 2544-2549.(Link)