Given the time intensive nature of gene engineering, the relatively straightforward and quick (<1 week) process of guide RNA validation can save weeks of cell culture and hours of bench time and ensure you're work with the most optimized sgRNA available. Further to this, a clear idea of gRNA activity will provide insights into how many clones need to be screened to identify a positive.
sgRNA Validation Methodologies
There are now a variety of methodologies available to scientists looking to compare and validate guide RNA activity. Below we detail some of the most commonly used.
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1. Mismatch cleavage assay
These assays rely on cleavage of a mismatched PCR product as a proxy for the amount of disruption that has occurred in the genomic DNA of a pool of cells. The more cleavage means more disruption by Cas9 at the target site.
There are currently two commercially available and widely used assays. At Horizon we use the SURVEYOR® assay from IDT, either in the cell line to be targeted, or in our workhorse line for validation - HEK293T. Thermo also offers the similar GeneArt® Genomic Cleavage Detection Kit.
To assess Cas9 cutting at a specific locus using a SURVEYOR® assay, PCR primers are designed to produce a PCR amplicon of between 250 to 800 bp. The primers are designed such that the target cut region is located at approximately one third of the length of the amplicon (from either end). This will enable two distinct products to be resolved on a polyacrylamide or agarose gel and the efficiency of cutting to be assessed.
In order to achieve consistent and sensitive Surveyor results, there are various steps in the experimental process that can be optimised. Here’s what we’ve found works well for us:
Tips for better Surveyor assay results
- Optimal PCR product size is 500 bp (no bigger than 800 bp)
- Location of target cut site should be approximately one third away from the product ends
PCR Set up
- PCR product must be clean with no non-specific bands present. A touch-down PCR based approach can help to generate a clean product for the assay.
- Purified PCR product should be at a concentration of 150 ng/µL
- Sequence PCR product to ensure that there are no SNPs that might alter expected results
- Horizon uses 5x Colorless GoTaq® buffer during hybridisation
- Hybridise in a 10 µL volume
- Use 400 ng of PCR product in the hybridisation reaction (ideally this should be approximately 2 µL of purified PCR product and 8 µL of mastermix made up as follows)
Reaction size ( ml) 10 Vol of template ( ml) 2 no of reactions 1 H20 4.7 MgCl2 (25 mM) 0.6 DMSO 0.6 5x Clear Gotaq Buffer 2 dNTPs (25mM) 0.1 volume (- template) 8
- Hybridise the PCR products in a thermocycler
- Keep Surveyor nuclease on ice at all times (only remove from the freezer immediately prior to use and return immediately after use).
- A mastermix of Surveyor nuclease and enhancer can be prepared if lots of samples are being assessed.
- Add Surveyor nuclease and enhancer directly to the 10 µL hybridisation reaction
- Digest the PCR products at 42ºC in a thermocycler
- Assess digest products on a gel (2% agarose or polyacrylamide) immediately post incubation. Note: PAGE gels often improve the resolution of the digest products
2. Sequence trace decomposition analysis
This approach relies on a specially developed algorithm to analyse Sanger sequencing trace and accurately determine the frequency of mutations at the projected editing site in a cell population down to 1-2%. This approach was published by Brinkman et al in Nucleic Acid Research and the tool (named TIDE) made available on a free to use web portal.
3. Indel Detection by Amplicon Analysis (IDAA)
If you have access to DNA capillary electrophoresis detection such as the ABI3010 sequenator then Yang et al’s IDAA method, which is based on tri-primer amplicon labelling, is both more sensitive than Surveyor and amenable to high throughput analysis.
4. Digital PCR
Surveyor assay is sensitive to about 5% cutting efficiency – which means that as long as 1 in 20 cells in your pool has undergone modification you should detect it. For many cell lines however this will be insufficient.
Digital PCR offers not only a more sensitive test, but a much more quantitative one, and you are literally counting the number of non-modified versus modified amplicons.
There are various ways to use digital PCR to quantify targeting efficiency, which vary in the design of the hydrolysis assay used.
One example is a single PCR amplicon spanning the target site, one fluorophore labelled hydrolysis probe that will bind every amplicon (thus quantifying total PCR product) and a differentially labelled probe that will bind the at the unmodified target site. In this manner, % modified amplicons can be extrapolated and efficiency quantified.
5. Immunofluorescence analysis
If you have a good, validated antibody against the protein that you’re aiming to knockout with CRISPR, then immunofluorescence microscopy or flow cytometry can represent rapid, quantitative methods to compare guide activity.
In the case of microscopy, cells can be seeded onto coverslips, transfected with the gRNA/Cas9 plasmid, and 72-96h later immuno-stained and counted.
This approach has the added advantage of quantifying protein knockouts, whereas methods above quantify mutations even if they do not cause frameshifts.
6. Clonal analysis of sgRNA targeted cells
If you are working in a highly optimised system, then skipping the validation step and moving straight to a single cell plate out can be relatively low risk.
For example, for our Hap1 knockout projects at Horizon we expect targeting efficiencies of >40% (following positive selection of transfected cells). As such we move straight to clonal analysis, by PCR and sequencing, which not only allows us to assess guide activity, but gives us our final product – knockout clones.
Prefer to skip sgRNA validation altogether?
Finally, this all important step can be skipped by purchasing a ready-made knockout cell line. Horizon's portfolio of 9000 knockout cell lines allows you to quickly bring CRISPR gene editing technology to your research, without having to spend time and resource doing this yourself. The money back guarantee also allows you to test the cell line with confidence.
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