Developed from an endogenous bacterial immune system, the gene-editing tool CRISPR exploded into the biological space in 2013 and now seems to be here to stay. The number of publications using the technique has increased exponentially in recent years—CRISPR gene editing is now performed in a wide variety of cell types, research areas, and for a myriad of applications, from creation of transgenic animals, and whole genome screening, to editing of single bases. The inherent simplicity of CRISPR has no doubt contributed to its widespread and rapid adoption, as gene editing can be performed in almost any cell type that is amenable to transfection. CRISPR gene editing requires only two components—a Cas nuclease and a short guide RNA (sgRNA) to guide it to the target site—which are delivered using standard molecular biology techniques.

But conducting a CRISPR experiment is not a trivial undertaking—experiments may require significant optimization, which can be time consuming and labor intensive. It has been estimated that a single CRISPR experiment takes on average 10 weeks to perform—and that’s assuming success on the first try, with experiments usually restarted around 7 times.1 In this article, we look at the most challenging aspects of the CRISPR workflow and some potential solutions that CRISPR researchers can adopt to improve efficiency and generate results they are confident in.

Tips on how to improve your CRISPR experiment

CRISPR workflows are relatively simple but can be time consuming and labor intensive. However, there are several things researchers can do to improve their CRISPR experiments and potentially save time and effort:

  • Design: use computational algorithms to select the most efficient sgRNA that will result in robust and complete knockdown with minimal off-target cleavage, or use commercially available, pre-validated guides for your experiment
  • Deliver: optimize the transfection protocol for your particular experiment to ensure optimal delivery and editing, which will save time in downstream steps—alternatively, consider outsourcing to a CRO
  • Detect: validate your edit to make sure it is correct—both at the genomic and proteomic level so you can be confident in your results and subsequent conclusions.

Good sgRNA design is key

One of the first things to do when embarking on a CRISPR experiment is to design the sgRNA with which to target your gene of interest. The most commonly used Cas nuclease is the Cas9 from S. pyogenes, which binds the 3’-NGG-5’ PAM sequence (Protospacer Adjacent Motif) that is present throughout the genome. As long as the 20-nucleotide sequence immediately adjacent to the PAM site matches the sgRNA, then the nuclease activity of Cas will be activated, causing a double-strand break at the chosen target site. CRISPR-Cas9 gene editing then harnesses the non-homologous end joining (NHEJ) DNA repair pathway, and the introduction of INDELs at the target site results in robust knockout of the target gene. Expansion of the CRISPR toolkit with modified or alternate Cas proteins means a range of edits are also possible, from regulation of gene expression with CRISPRi and CRISPRa, to base editing, and even RNA editing.

A significant challenge when selecting sgRNA is ensuring that your chosen target doesn’t contain significant homology to sites elsewhere in the genome—otherwise you’ll be at risk of off-target cleavage events, which could have unintended cellular consequences. But it’s also important to consider the position of your edit and subsequent INDEL—a recent paper by Tuladhar et al suggested that some INDELs can still result in expression of protein products due to alterations to the mRNA, including activation of translational reinitiation or exon skipping.2 It’s good practice to include several different guides per gene in your experiment to avoid having to start all over again if it turns out your chosen sgRNA is inefficient. There are several computational methods available that can be used to predict the best and most efficient sgRNA to use in your particular experiment—or you could consider using a commercially available sgRNA that has already been pre-validated to ensure optimal results.

Deliver CRISPR

Once you have your CRISPR components, they then need to be delivered into the cell so that gene editing can occur. This is a crucial step in any CRISPR workflow as the efficiency of delivery, and the related editing, can have a profound effect on the time taken to complete the experiment. CRISPR components can be delivered by plasmid, via lentivirus, or as pre-complexed ribonucleoprotein (RNP), either by chemical (e.g. lipofection) or physical means (electroporation, microinjection), or by viral vector. There are advantages and disadvantages of each transfection method, and the choice is dependent upon a number of factors, including the type of expression required for the experiment (stable versus transient expression) and the cell type you are working with. One way to save time is to use commercially available reagents—and in the case of using RNP, you can also skip the cloning steps involved in generation of expression plasmids.

Whichever method and format of CRISPR components you select, it will require significant optimization to ensure optimal delivery and editing efficiency—but it will be worth it. “It’s all a numbers game,” says Dr Anthony Adamson, Manager of the Genome Editing Unit at the University of Manchester, U.K., a core facility that offers assistance to researchers looking to use CRISPR. “The better your delivery and editing efficiency, the fewer clones that need to be screened to find your edited cells. A bit of extra effort upstream helps with this inevitable bottleneck”. Core facilities at universities or research institutes, or Contract Research Organizations (CRO) offer gene editing as a service and can help with the sometimes-time-consuming optimization stage—as well as experimental design, analysis, or indeed, the whole gene-editing process.

Check your edit

So you have successfully delivered your CRISPR components and selected your positive cells. But do you have the right edit? Validation is essential to ensure confidence in your subsequent data and conclusions. Methods such as PCR amplification or mismatch cleavage assays for INDEL detection are relatively quick and simple ways to check your edit—particularly useful in the optimization phase of your CRISPR experiment. When more detailed genotyping is required, the gold standard validation tool is next-generation sequencing – but if the cost of NGS is prohibitive, there are alternative NGS-based validation methods, such as CIRCLE-Seq and DISCOVER-Seq that have been developed.3,4

It is also important to consider the effect of your edit on protein expression, and that the edit you have made is truly representative of gene loss. A recent Nature paper looked at 193 genetically verified deletions and found that one-third displayed residual protein expression,5 and so edited cells may still be able to express mutated or truncated proteins that retain some functional capacity. Adding a proteomic validation step to your workflow will give added confidence to your findings, and any subsequent experiments. But if you are using an antibody in your validation step, remember to consider the potential effect of your edit— for example, the edit could ablate the antibody epitope but retain a functional protein.

CRISPR: DIY or outsource?

The speed and ease of CRISPR has moved gene editing to within the capabilities of most scientists with good molecular and cell biology skills. But CRISPR can take time to master, which can be a significant time and cost investment at odds with researchers having to meet project deadlines and maintain publications. Investing time and/or money in your CRISPR experiments, by rigorously optimizing the process, by using commercially available, pre-validated CRISPR reagents, or by outsourcing some or all of the gene-editing process will no doubt reap rewards in efficiency and quality of results generated.

References

1. Synthego benchmark report

2. Tuladhar, R. et al. CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. Nature Communications10, (2019).

3. Tsai, S. Q. et al. CIRCLE-seq: A highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nature Methods 14, 607–614 (2017).

4. Wienert, B. et al. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. Science364, 286–289 (2019).

5. Smits, A. H. et al. Biological plasticity rescues target activity in CRISPR knock outs. Nature Methods 16, 1087–1093 (2019).