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How to Validate a CRISPR Experiment  

Content brought to you by Sigma-Aldrich® Advanced Genomics

Image of sequencing data to depict validation of successful CRISPR gene editing.

As with any research experiment, it’s vital that you validate your CRISPR-driven gene editing. At a minimum, you will need to confirm:

  1. Delivery of the CRISPR reagents into your cells.
  2. Successful and specific editing of the target genes.
  3. The expected change of expression of the protein encoded by the target gene.

This article provides an introduction to the general methods and controls most widely employed for validating a CRISPR experiment.

Validating Delivery of CRISPR Reagents

Broadly, there are two popular methods used to validate the successful delivery of CRISPR components: 1) fluorophore expression and 2) antibiotic resistance. Each method has unique characteristics and you may choose to use them simultaneously.

Validating CRISPR Reagent Delivery by Fluorophore Expression

Fluorophore expression provides a simple, yet colorful, way to check if your CRISPR reagents have been successfully delivered into your cells. Fluorophore expression can be achieved by using vectors that express a fluorophore such as green fluorescent protein (GFP) if using plasmid- or lentiviral-based delivery methods. For RNP complex delivery, you can use a guide RNA (RNA) or nuclease tagged with a fluorophore. In both cases, delivery is confirmed by the presence of the fluorophore in your cells using either fluorescence-activated cell sorting (FACS) or microscopy.

Fluorophore expression also allows you to calculate the efficiency of the delivery method by comparing the number of cells expressing the fluorophore to the total number of cells in the sample. If using FACS, sample enrichment is possible by isolating only those cells that express the highest level of the fluorophore.

MilliporeSigma has recombinant Cas9-GFP proteins (RNP complexes) and several different vectors for plasmid transfection or lentiviral transduction that express a variety of fluorophores. These CRISPR controls allow you to determine if delivery of the CRISPR reagents into your cell was successful and monitor their clearance from the cell in real-time by fluorescence-activated cell sorting (FACS). [1]

Validating CRISPR Reagent Delivery by Antibiotic Selection

In addition to expressing fluorophores such as GFP, some vectors contain an antibiotic resistance gene such as Puromycin-N-acetyltransferase (pac). With an antibiotic selection marker, the survival of cells in the presence of the antibiotic provides validation of the delivery of plasmids and subsequent expression of the encoded nucleases. Antibiotic selection also makes it possible to isolate and enrich only those cells that have incorporated the CRISPR components.

It is important to note that although fluorophore expression or antibiotic selection can only confirm successful CRISPR reagent delivery they do not determine if the desired sequence was successfully targeted.

Validating Successful Genetic Targeting

Confirmation of successful gene targeting requires the detection of the insertions or deletions (indels) introduced by the CRISPR experiment. [2] Here we describe a few of the most common methods, but your choice will depend on a range of factors, including the type of gene edits you wish to make and your budget.

Sanger DNA Sequencing

Sanger DNA sequencing remains the gold standard for validation owing to its reliability, sensitivity, and its ability to precisely identify mutations introduced. The primary drawback to Sanger sequencing is the time-consuming and labor-intensive nature of the method; multiple preparation steps are required to establish a clonal cell population before sequencing.

Next-Generation Sequencing

Next-generation sequencing (NGS) has the distinct advantage of bypassing the need to establish a clonal cell population harboring the mutation. NGS can identify even rare mutations in a cell sub-population. Although mandatory in cases of animal and human applications, the error rate and cost per run remain high for NGS approaches. The continued advancement of sequencing technology and falling costs suggests that NGS will soon become the dominant validation option for CRISPR editing.

Surveyor Nuclease Assay

The Surveyor™ nuclease assay is an accessible method for researchers and is based on the principle of mismatch detection. [3] In this method, a pool of PCR products from the edited alleles is denatured and rehybridized, after which the Surveyor nuclease selectively detects and cuts those pairs that are mismatched due to the presence of an indel on one of the strands introduced by the cellular NHEJ repair process. Cleaved DNA amplicons that incorporate a mismatch are detected on an agarose gel. Further confirmation that mutations have been introduced at the cleavage site can be achieved with Sanger sequencing.

The Surveyor nuclease assay is a fast and affordable method, but it has limitations. For example, the assay will miss some small indels and it is not able to distinguish CRISPR-derived indels from naturally occurring polymorphisms. [4]

TIDE Assay

In cases where a more sensitive indel detection method is needed, the Tracking of Indels by Decomposition (TIDE), may be more suitable. TIDE is a three-step method where the genomic region targeted by the nuclease is amplified from DNA isolated from transfected cells. Sanger sequences of PCR products are analyzed by software to determine if indels are present based on comparison with the wild-type sequence. The software also identifies precise break sites and estimates the statistical significance of each indel. TIDE reduces the total cost of validation, as it allows Sanger sequencing to be performed on mixed cell populations. However, this pooled approach means that it cannot distinguish two alleles of the same length, and it struggles with rare alleles. Moreover, the reliability of TIDE is dependent on the quality of the PCR products and Sanger sequences.

Validating Successful Loss of Expression

The successful introduction of an indel mutation into your gene of interest does not guarantee the disruption of expression of the corresponding protein. It is therefore important to confirm that the protein encoded by the target gene is no longer expressed in your edited cells.

The simplest way to check for protein expression is by western blot using a well-validated antibody.  When possible, it is preferential to select an antibody that recognizes an epitope towards the N-terminus of the expected indel to detect if a truncated form of the protein is being expressed.

Experimental Controls for Gene-Editing Experiments

As with any other experimental design, consideration should be paid to ensure strong controls are in place. Controls should be included with each experiment, run in parallel, and with the same reagents. The two fundamental controls for a gene-editing experiment can be grouped as positive and negative.

Negative Controls

A negative control demonstrates that the observed change is a direct result of the introduced mutation and not due to other non-specific effects. A typical negative control consists of all the necessary CRISPR reagents to perform gene editing but uses a gRNA that does not recognize any sequence in your experimental system. Off the shelf negative controls that have been designed not to target any region in the human, rat, or mouse genome are available from the Sigma-Aldrich® portfolio.

Positive Controls

A positive control demonstrates that the CRISPR reagents and delivery method used are working as expected in your experimental setup. This is important in cases where the experiment produces a negative result (or no observed change), as it allows you to confirm this is not due to your experimental design. A typical positive control consists of all the necessary CRISPR reagents required to perform gene editing along with a gRNA that has previously been shown to successfully target another gene in your system. We offer a range of validated controls in a variety of delivery formats that can be used to validate your CRISPR system.

CRISPR Validation Summarized

A well-conceived CRISPR experiment includes a strong plan for validation and controls. Proper validation of both gene editing and loss of expression will significantly improve your ability to identify potential problems and correct, as necessary.

More CRISPR Resources

  1. CRISPR Gene-editing: Considerations and Getting Started (Article)
  2. How to Understand CRISPR Formats and Their Applications (Article)
  3. Engineering Vero Cell Lines Using CRISPR to Increase Production of Viral Vaccines (Webinar)
  4. CRISPR Gene Editing 101 (eBook – coming soon)

And you should also consider the following Sigma-Aldrich® resources:

  1. CRISPR Lentiviral Formats
  2. CRISPR Controls
  3. CRISPR Essentials
  4. Quick ordering
  5. Advanced Genomics Resource Center
  6. Sigma-Aldrich® Advanced Genomics
  7. Additional CRISPR Webinars

References

  1. Oh SA, et alRibonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells. Curr Protoc Immunol. 124 (1): e69 (2019). DOI: 10.1002/cpim.69. Epub 2018 Oct 18.
  2. Kosicki, M. et al. Dynamics of indel profiles induced by various CRISPR/Cas9 delivery methods. Prog Mol Biol Transl Sci. 152, 49–67 (2017). DOI: 10.1016/bs.pmbts.2017.09.003
  3. Qiu, P. et al. Mutation detection using Surveyor™ nuclease. Biotechniques, 36(4), 702-7 (2004). DOI: 10.2144/04364PF01
  4. Germini, D, et al. A comparison of techniques to evaluate the effectiveness of genome editing. Trends Biotech. 36 (2), 147-159 (2018). DOI: 10.1016/j.tibtech.2017.10.008
Image of sequencing data to depict validation of successful CRISPR gene editing.

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