Common CRISPR pitfalls and how to avoid them

With rapidly increasing amounts of information, various reagent formats, and numerous applications, where does one start when planning a gene-editing experiment? What do you need to think about when choosing your biological system? What reagents are suitable for your experimental aim and constraints?

Here we discuss some points, that when considered in advance, will make your CRISPR-based gene editing experiments more successful.

Note: This article was originally published in June 2020, but has been updated and republished Dec. 2021.

Choose your cell line wisely

image for crispr pitfall blog post

When choosing a suitable cell model that reflects the biology of what you are studying, consider how your choice will impact your gene editing options.

Ideally, your cell line of choice has been well genotyped and characterized. In addition, it should have low gene copy numbers, can be cultured for an infinite amount of passages, and is easily transfectable. These properties relate to the choice of CRISPR reagent formats and the efficiency of gene editing events.

If you are diluting the cells out to single-cell isolation, cell lines must be able to be grown from a single cell and, ideally, do so rapidly. Cell lines with a high growth velocity simplify the clonal selection by reducing the time needed to grow up the clones.

Consider the impact of your desired mutation on cell biology

What is the impact of your desired gene knockout on cell viability and growth? Mutations that impact viability or growth will impact the extent of screening that you need to perform to find your desired knockout, and the duration of the experiment. Essential genes will not allow for a complete knockout, you may likely observe truncated, but still functional proteins or heterozygous mutations. It may still be worthwhile attempting the edit, or trying a less-penetrant knockdown strategy, like siRNA.

Understand your gene’s sequence variability to identify functional guide RNAs

Obtaining reliable results depends on knowledge of transcript related variants and any SNPs in your cell line of choice. Information on reference sequences can be found in Ensembl and NCBI databases, or you can even sequence your target locus to be sure. Guide RNAs should be designed against the most relevant transcript isoform(s), target all transcripts of interest, and exclude any SNPs. Make sure to check the specificity of any self-made designs to identify all off-target alignments.

For standard CRISPR projects, you can rely on our predesigned synthetic, or lentiviral guide RNA designs targeting all human and mouse NCBI-annotated genes. Take advantage of our algorithm for functional, highly-specific, predesigned guide RNAs, which are guaranteed to edit your gene of interest. All catalog synthetic guides carry stabilizing modifications that improve editing efficiency in DNA-free applications that require protection from cellular systems with high nuclease activity.

If our predesigned catalog doesn’t work for your application, use the CRISPR Design Tool to customize your designs. The tool offers quick and easy design for gene knockout in over 30 other species, supports non-S.pyogenes Cas9 nucleases designs, and generates guides that edit a custom-defined region of the genome (e.g., for knock-in experiments). The design tool comes with a rigorous specificity check and allows you to target either protein-coding, microRNA, or lncRNA genes.

Select appropriate reagent formats and optimize delivery conditions

Delivery of CRISPR reagents into your cells is one of the most critical variables in generating high editing efficiencies. Transfection optimization variables are focused around cell density and the amount of lipofection reagent. Forward transfection is where the transfection reagents are added on top of previously plated cells; this works for cell types that adhere to plates or wells. However, if your experiment requires you to work with cells in suspension or when screening in a high-throughput format, reverse transfection, where cells are added to the plated transfection reagent complex, is the preferred method.

For any challenging to transfect cell type, lentiviral delivery is a great alternative. For lentiviral delivery, the most critical steps to be aware of are transduction efficiency, and to make sure to choose a promoter with high activity in your cell line of choice.

It is also critical to include CRISPR controls in your assay. The best way to figure out what experimental conditions will result in high efficiency of gene editing in your cells is to use a positive control guide RNA. Positive controls help to work out critical experimental variables such as transfection/transduction conditions, reagent concentration, and timing parameters.

It is important to note that you likely will not see 100% editing efficiency: Under optimized delivery conditions, about 25-75% of cells will carry your desired edit.

This article will help you choose the Cas9 nuclease format appropriate for your experimental needs.

Enlist enrichment strategies to streamline edited cell selection

Even if the biology of your system limits your delivery options, and your editing efficiency or knockout viability is low, there are ways to find cells with your desired edit. One option is just to screen more clones to find that needle in the haystack edit. Another option is to use a selection strategy, such as FACS sorting (based on fluorescent reporters or gene-specific cell surface markers) or antibiotic selection. These are great ways to enrich for positive clones and limit the number of clones you must screen to identify a positive one. No matter which delivery method you choose, we offer adapted Cas9 protocols and products for such enrichment methods, such as fluorescent or antibiotic-resistant lentiviral Cas9 and sgRNA reagents, and DNA-free options like fluorescent Cas9 mRNA.

Boost HDR efficiency for knock-in experiments

HDR efficiency can be improved by designing your donor in a way that the original CRISPR PAM site is edited with a silent mutation, which avoids re-cleavage of the target site. HDR requires a donor to be present in addition to the site-specific guide RNA, and Cas9 nuclease, donor format choice (e.g., ssDNA or plasmid), as well as homology arm length, can have effects on the efficiency of knock-in events.

Learn about further HDR optimization strategies in this poster.

Choose an appropriate validation strategy

Similar to reagent format choice, and delivery method of the reagents, the validation strategy will be dependent on the cell type and downstream application. If you are working on locking down a protocol, you may start by getting an idea of editing efficiency by running a T7E1 gel. This analysis method is a cost-effective and straightforward technique but should only be used as a ballpark estimate as it tends to under represent the actual editing efficiency.

If you are attempting to generate a functional protein knockout, you are likely going to need to look at some type of antibody-related validation. Validation for this type of edit can come in the way of running a Western and looking at the disappearance of a band or using immunofluorescence to look at wild type versus treated cells. These validation techniques need to be refined for your specific experiment as even when a protein is functionally knocked out a band can still show up on a Western if the antibody is binding to a non-functional domain.

Lastly, if you are working on developing a cell model that includes a knock-in, sequencing multiple clones will be a required validation step. Unfortunately, there isn’t a way around sequencing to find clones that have incorporated your insert of interest. Therefore it is critical to use a guide RNA and transfection method that gives you the highest potential editing efficiency to reduce your sequencing burden.

Commonly used assays to detect and validate your CRISPR edit are found here.


Ryan Donnelly headshotWritten by Ryan Donnelly

He holds a Professional Science Masters in Molecular Biotechnology from George Washington University and Bachelors in Chemistry from the University of Florida.




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