Plan Your CRISPR Experiment

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful system that enables researchers to manipulate the genome with relative ease. Over the past decade, researchers have expanded the CRISPR toolbox to include many different types of edits, including point mutations, base changes, and large insertions or deletions. For more information on the wide range of CRISPR technologies, visit our CRISPR Guide.

There are many different Cas proteins available, and researchers can adapt these experiments for different organisms. This page provides a general framework to get you started using CRISPR in your research and setting up CRISPR experiments. We will focus on making single edits using CRISPR/Cas9 in mammalian cells as an example, but many of these principles also apply to using CRISPR in other organisms.

As with most experiments, you first need to have a biological question in mind. Then you can decide how to answer that question using genetic manipulation to model a specific disease or process of interest. Do you want to:

• Generate complete and permanent loss of gene expression or function (knockout)?
• Generate a specific mutant allele of a gene (point mutant)?
• Increase or decrease expression of a target gene?

Once you have a clear understanding of your experimental goal, you are ready to start designing the specific components (Cas enzyme and guide RNA) for your experiment (Figure 1). You will decide how to express Cas9, the delivery system for Cas9 and the guide RNA (gRNA), the design of the gRNA sequence, and finally how to validate your genetic edit.

Figure 1: Flow chart describing the general framework of a CRISPR experiment. Created with BioRender.com.

Select Your Desired Genetic Manipulation

Different genetic manipulations require different CRISPR components. Selecting the genetic manipulation you plan to perform will help you narrow down which components you’ll need for a given experiment. In the table below, we highlight the most common categories of genetic manipulations used in mammalian cells and their corresponding Cas enzymes. In the next section, we will cover strategies for designing the gRNA component for each category.

Genetic Manipulation	Application	Cas Enzyme	Additional Considerations	Browse Plasmids
Knockout	Permanently disrupt gene function; cells will use error-prone non-homologous end joining (NHEJ) at the cut site	Cas9 or Cas9 nickase	High-fidelity Cas enzymes increase specificity Dual-nickase approach increases specificity but is less efficient Each putative knockout allele must be experimentally verified	Cut Nick
Homology Directed Repair (HDR)	Generate specific genomic edits, like point mutations or small tag insertions, by copying from a DNA donor template	Cas9 or Cas9 fusions with proteins that promote HDR	Requires a repair template Lower editing efficiency compared to NHEJ knockout	Cut Nick
Base Edit	Generate specific base pair edits without double-strand breaks	Base editor (dCas9 or Cas9 nickase fusions with different base editing proteins)	Many base editors fused to Cas9 exert their activity within a very narrow window proximal to the PAM sequence	Base Edit
Prime Edit	Generate targeted insertions, deletions, and point mutations using a prime editing guide RNA (pegRNA)	Prime editor (Cas9 nickase fused to reverse transcriptase (RT))	Can edit within a wider window than most base editors Editing efficiency can be low, but generally higher than HDR pegRNA optimization can be challenging	Prime Edit
Repress or Interfere (CRISPRi)	Reduce gene expression without permanently modifying the genome sequence	dCas9 or dCas9 fusion with transcriptional repressor (such as dCas9-KRAB)	dCas9-KRAB can be more effective than dCas9 alone for mammalian cell lines	Interfere
Activate (CRISPRa)	Increase gene expression without permanently modifying the genome sequence	dCas9 fusion with transcriptional activator (such as dCas9-VP64)	Many different activator systems exist, including the multi-plasmid SAM system	Activate

Design and Clone Your gRNA

Achieving your desired edit comes down to location, location, location. Positioning your chosen Cas enzyme at the right spot within the genome relies on proper gRNA design. While there are some cases where it makes sense to manually design a gRNA, in most cases gRNA design software is better and more efficient at designing potential gRNAs for you to choose from. Below are a few factors to keep in mind when designing and cloning your gRNAs.

Select Genetic Element to be Targeted

The exact region of the gene you target will depend on your specific application.

• Knockout: Target constitutively expressed regions, 5’ exons, or an exon known to code for an essential protein domain.
  • Targeting a 5’ exon reduces the chances that the targeted region is removed from the mRNA due to alternative splicing, and an early frameshift mutation is more likely to result in a non-functional protein product. Be aware that there could be downstream translation start sites that could allow for expression of a truncated protein.
  • Targeting an exon coding for an essential protein domain could abolish protein activity and essentially function as a “knockout,” but be sure to consider potential consequences of having a non-functional form of your protein expressed in your system.
• HDR: Select a cut site as close as possible to the location of the desired edit, ideally less than 10 bp away. For more information, read our three tips to improve HDR efficiency.
  • Remember that HDR also requires donor DNA to serve as template for the repair. Once you settle on your gRNA, you can design a donor DNA to have the appropriate left and right homology arms. See Addgene's HDR blog post for more details.
  • In some cases, there may be no PAM site close to your desired edit site. Consider alternative Cas9 enzymes with different PAM sequences or Cas9 enzymes with flexible PAM sites.
• Base Editing: Target the site of your desired edit. Many base editors exert their functions within a limited window from the PAM sequence, and base editor variants often have different optimal editing windows. Be sure to refer to the literature associated with your chosen editor.
• Prime Editing: pegRNAs must serve as both a guide and a template. In general, edits must be downstream (3’) of the nick site, and guides that target closer to the edit site yield higher editing efficiencies.
  • The template sequences can also have a large impact on editing efficiency. So while testing multiple gRNAs is a good idea for most CRISPR experiments, testing multiple pegRNA designs is particularly important for prime editing.
• CRISPR Intereference: Target the promoter region of the gene of interest, where the Cas9 protein can block transcription from ever initiating. gRNAs targeting regions within the gene itself can also be effective, as the Cas9 bound to the DNA can interfere with transcription elongation.
• CRISPR Activation: Target the transcription start site. CRISPRa requires dCas9 to be fused to transcriptional activators that need to be within a given range of the transcription start site in order to function effectively.

Select gRNAs Based On Predicted On-Target and Off-Target Activity

Another important factor to keep in mind when designing your gRNA is the balance between on-target and off-target activity. In a perfect world, your gRNA sequence would be an exact complement to your target sequence with no homologous sites elsewhere in the genome. In reality, a given gRNA target sequence will likely be able to bind to additional sites throughout the genome. These sites are called off-targets. Be aware of off-target sites when designing and selecting a gRNA, but you likely won’t be able to completely avoid them. To increase specificity, consider using an engineered Cas enzyme with higher fidelity.

In addition to off-target activity, it is also important to consider factors that maximize cleavage of the desired target sequence, or on-target activity. Two gRNA targeting sequences with 100% homology to their DNA targets may not result in equivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides within the selected target sequence. Additionally, any mismatches between a given gRNA sequence and your genomic target may reduce the gRNA activity. Be sure to sequence the genomic region you intend to target in your samples to ensure there are no discrepancies with the reference sequence you used for gRNA design.

Addgene offers many plasmids containing validated gRNAs. These plasmids contain gRNAs that have been used successfully in genome engineering experiments. Using validated gRNAs can save your lab valuable time and resources when carrying out CRISPR experiments and can be used as positive controls when working out a new protocol.

Read more about how to design your gRNA.

Synthesize and Clone Desired gRNAs

Once your target sequences are selected, it’s time to design your gRNA oligos and clone them into your desired vector. In many cases, you can synthesize, anneal, and insert the targeting oligos into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA vector you have chosen, so it is best to review the protocol associated with the specific plasmid in question (see CRISPR protocols from Addgene depositors).

Browse empty gRNA expression vectors.

Select Your Expression and Delivery System

Once you have selected your target and designed your gRNA(s), the next step is to choose how to express your Cas enzyme and gRNA(s) in your target cells. There are a variety of ways to deliver and express these components (Figure 2), and the optimal system will likely depend on your cell type.

Figure 2: Different delivery systems utilized for CRISPR/Cas gene editing. This image summarizes ways that the CRISPR/Cas system can be delivered to cells. The system may be delivered using viral methods, as DNA or mRNA encoding the Cas enzyme and gRNA, or as a functional ribonucleoprotein (RNP). Created with BioRender.com.

When choosing your system, consider which Cas enzyme you are using (as species and size may affect what will work for your experiment) and the expression time (transient or stable). For plasmid and viral-based approaches, the species and expression pattern of the promoter for both your Cas enzyme and gRNA and the presence of a selectable marker (antibiotic resistance or fluorophore) are also relevant.

Delivery Using Plasmids

The most straightforward method to deliver a CRISPR system into mammalian cells is to directly transfect expression plasmids encoding your Cas protein and gRNA, which you generally perform by chemical transfection or electroporation. Expression can be transient, or you can generate stable cell lines if your plasmid contains a selection marker. Plasmid delivery is versatile thanks to the wide range of options for the Cas enzyme, promoters, and selection markers. There are also no packaging limits as there are for viral vectors — the only size limitation is what you can efficiently transfect. However, note that this method is most useful for cell lines that you can transfect at high efficiency, such as human embryonic kidney 293 (HEK293) cells. For more difficult cell types, you may need to look into other options.

Browse Addgene's collection of CRISPR plasmids.

Delivery Using Viral Vectors

Viral vectors are a great option, as they offer high expression levels, long-term and stable expression, are safe and easy to work with, and are good choices for more difficult cell types like primary cells. For CRISPR delivery, lentiviral and adeno-associated virus (AAV) vectors are the most common.

Using viral vectors takes a bit more prep work, as you first have to produce the viral particles. Then you transduce your cells using the viral particles before you continue to selection, validation, or other downstream methods. For more information about viral vectors and their production, see our viral vector guides.

Lentiviral vectors

Lentiviral vectors are a popular option for producing stable cell lines, since the genetic cargo (in this case, CRISPR components) integrates into the genome. Lentiviral vectors are a good option for difficult-to-transfect cells. You can direct expression to specific cell types by using cell-specific promoters, which is useful for in vivo studies. While we don’t discuss CRISPR screens in this guide, lentiviral vectors are a common choice for conducting genome-wide screens using CRISPR.

Browse Addgene's collection of in-stock lentiviral preps.

AAV vectors

AAV vectors offer some advantages for viral vector delivery. Both lentiviral and AAV vectors are safe to use in the lab, but AAVs exhibits the lowest immunogenicity and are one of the least toxic methods for in vivo viral delivery. Additionally, transgene expression with AAVs can be long-lived, despite recombinant AAVs not integrating into the host genome. Scientists also frequently use AAVs to develop CRISPR-based therapeutics.

One limitation of AAV vectors is that the packaging limit (how large of a DNA sequence you can actually package into your viral vector) is smaller than that of lentivirus. Thus, AAVs are generally only compatible with smaller Cas enzymes, like S. aureus Cas9. For more information, see our blog post on overcoming the AAV size limitation.

Browse Addgene's in-stock AAV preps or try Addgene's Packaged on Request service.

Delivery Using mRNA and Proteins

In addition to the methods described above, you can also directly deliver mature mRNA or purified protein into cells. These methods are more transient than plasmid transfection or viral vectors, reducing the risk of accumulating off-target effects.

RNA delivery

The first of these methods is RNA delivery of Cas and gRNA. This method involves using in vitro transcription reactions to generate mature Cas mRNA and gRNA, which you can deliver to target cells through microinjection or electroporation. The target cells translate the mRNA encoding Cas to produce the Cas enzyme, and the enzyme then forms a complex with the gRNA for CRISPR activity. Because you are delivering the mRNA directly to the cells, there is only a short window of CRISPR activity since expression decreases as the RNA is degraded within the cell. However, this can be desirable to help decrease off-target effects. This method is a common choice for generating modified mouse lines.

Protein delivery

Lastly, you can deliver Cas-gRNA ribonucleoprotein (RNP) complexes directly into your target cells. In this case, your target cells are not transcribing DNA to mRNA, nor do they need to translate mRNA to protein. Instead, you directly combine purified Cas protein and in vitro transcribed gRNA to form a Cas-gRNA complex. You then delivery the RNP to cells using cationic lipids, electroporation, or an endosomal approach. Similar to the mRNA method described above, this method is also transient, as the RNPs will only be active until they are degraded. This method is helpful in cells that are difficult to transfect, transduce, or may not express common promoters.

Browse Cas9 plasmids for RNP delivery.

As mentioned above, CRISPR efficiency will vary based on the method of delivery and the cell type. Before proceeding, we recommend asking labmates/colleagues, searching the literature, or discussing with your PI to help pick which method may work best for your experiment. It may be necessary to optimize your delivery conditions, especially if you are using a difficult cell type. For more information, take a look at our collection of blog posts about CRISPR expression systems and delivery methods.

Validate Genetic Modification

Once you have successfully delivered the gRNA and Cas enzyme to your target cells, it is time to validate your genome edit. CRISPR editing produces several possible genotypes within the resulting cell population. Some cells may remain wild type due to either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells that do express both Cas9 and gRNA. Before choosing a validation method, ask yourself some questions:

• Does the resulting population need to be identical (clonal) or is a mixed population okay?
• Does the edit need to be homozygous?
• What type of modification is it?
• Are you modifying the genomic sequence or affecting RNA or protein expression?

For more information, check out our blog post on validating CRISPR modifications.

Cell Population

The first step of validation is determining the type of cell population you need for your end goal. A clonal population is grown from a single cell and will result in cells with identical genomes. If your end goal is to establish an edited cell line, for example, you will likely need a clonal population. Additionally, clonal populations are useful for determining if your edit is homozygous or heterozygous, as you can resolve differences between edited alleles.

A clonal population is not always required, and a mixed population can be sufficient for many experiments. A mixed population will contain cells with a variety of modifications and will likely contain a subset of wild type cells. If you are screening a variety of gRNAs or a new type of Cas enzyme, validating edits in a mixed population can provide you with enough information on aspects like cleavage success and efficiency.

Edit Type

The type of edit you are looking for will ultimately depend on which CRISPR method you utilized. If your edit affects genome sequence, your edited cells may be homozygous or heterozygous at your target locus. Furthermore, in cells containing two mutated alleles, each mutated allele may be different owing to the error-prone nature of NHEJ. In HDR, base editing, or prime editing experiments, most mutated alleles will not contain the desired edit. Even if you provided a homology template, a large percentage of DNA breaks will be repaired by NHEJ instead of incorporating your desired edit through HDR. Base and prime editing are relatively inefficient methods and often require screening a larger pool of cells to find one with your desired edit.

Lastly, you will need to know if your edit just affects the genomic sequence or if it also affects RNA and/or protein expression. Generating knockouts, CRISPR activation or interference, and some targeted edits can lead to changes in more than just the DNA sequence. If this is the case, you can confirm your edit using DNA-based methods like sequencing or digital PCR, RNA-based methods like qPCR, and/or protein-based methods like a Western blot.

Validation Methods

There are several common ways to verify that your cells contain your desired edit. Each of these methods has their own considerations. Which one to use will come down to the goal of your experiment, the type of edit, and resources available to your lab.

Method	CRISPR Edit Type	Additional Considerations
Mismatch cleavage assay	Knockouts Some point mutations Some insertions/deletions	Very inefficient Can't distinguish specific edits
PCR	Knockouts Large insertions/deletions Some prime and base edits	Can be paired with restriction enzymes for further customization Usually paired with a sequencing method Can’t provide specific sequence information alone
Digital PCR	Knockouts Insertions/deletions Prime and base edits HDR	Can distinguish between homozygous and heterozygous edits High sensitivity (good for rare edits)
Sanger sequencing	Knockouts Prime and base edits	Good for random mutations Often difficult to distinguish specific edits (unless homozygous and/or clonal population)
Next-generation sequencing	Knockouts Prime and base edits HDR	Can distinguish specific edits Good for rare edits Many options like RNA-sequencing, Perturb-seq, deep sequencing, etc.
qPCR	Knockouts Activation/interference	Needs good primer design
Western blot	Knockouts Activation/interference	Needs a good antibody

References

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Content last reviewed on 11 July 2025.