CRISPR-Cas9 technology has revolutionized genome editing by providing efficient tools for gene knockouts, knockins, knock-ups, knock-downs, and more. In addition to being an invaluable tool in labs around the world, the first CRISPR therapy received FDA approval in December 2023 with many more therapies on the horizon. New components and approaches are constantly being developed to fully utilize this elegant system, so designing effective CRISPR experiments for research or clinical use can be difficult. This article provides an overview of the CRISPR-Cas9 system and strategies to optimize CRISPR vector design for successful results.
Basics of the CRISPR system
The CRISPR-Cas9 system contains multiple components whose use depends on the desired application, vector system, and target cell type. However, all CRISPR methods contain two crucial components: the Cas9 endonuclease and guide RNA (gRNA). The gRNA, a short RNA sequence, directs Cas9 to the target site by binding to complementary DNA in the target genome. Cas9, an RNA-guided DNA nuclease, introduces double-stranded breaks (DSBs) at specific genomic locations, as long as the target sequence is adjacent to a short DNA motif known as the Protospacer Adjacent Motif (PAM). Once DSBs are introduced at target sites, one of two main DNA repair pathways are used by the cells to correct the break (Figure 1):
a) Non-Homologous End Joining (NHEJ): This most commonly-used pathway often introduces small insertions or deletions leading to frameshift mutations that disrupt protein-coding regions, and hence, protein production. While NHEJ facilitates the creation of gene knockouts, it lacks precision and produces a heterogeneous population of edited cells.
b) Homology Directed Repair (HDR): This mechanism uses a donor DNA template for more accurate repair. The use of a donor sequence also allows introduction of precise changes including directed point mutations and large fragment knockins and knockouts at the site of the DSB. Single-stranded oligonucleotides (ssODNs) can be used as templates for small insertions (< 60 bp), or double-stranded DNA (dsDNA) can be used for insertion of larger sequences (up to 4-5 kb), such as fluorescent tags.
Figure 1. Mechanisms of CRISPR-induced DNA repair.
As CRISPR applications have diversified, users must consider which Cas9 to use. While SpCas9, derived from Streptococcus pyogenes and codon-optimized for various species, is the most commonly used Cas9 variant, other popular variants are used for a variety of applications beyond gene knockout, as summarized in the table below:
| Cas Enzyme | Application | PAM Sequence | Key Considerations |
|---|
| SpCas9 | Knockout, knockin | NGG | - Creates DSBs
- High efficiency and versatility
- Well established protocols
- Clinical use
|
| SaCas9 | Knockout, knockin (popular in AAV delivery) | NNGRRT | - Smaller size (~3.3 kb)
- Ideal for packaging into vector systems that have limited cargo space (e.g. AAV vectors)
|
| Cas9(D10a) nickase | Knockout (via paired nicking to minimize off-target effects) | Typically NGG* | - Generates single-strand cuts; DSBs through paired nicking
- Reduced off-target effects
- Requires design of two gRNAs, complicating the overall design
|
| Prime editors | Precise editing (insertions, deletions, point mutations) | Typically NGG* | - Uses a modified Cas9 fused with a reverse transcriptase to introduce precise edits without creating DSBs
- More complex design
- Less efficient for large fragments
|
Base editors (e.g. BE3) | Point mutation (direct base conversion) | Typically NGG* | - Enables conversion of one DNA base to another without introducing DSBs
- Limited to specific base transitions
|
| dCas9/VPR | Gene regulation (activation) | Typically NGG* | - Lacks nuclease activity, allowing modulation of gene expression without DNA cleavage
- Cannot produce permanent edits (e.g., knockouts or knockins)
|
| dCas9/KRAB/MeCP2 | Gene regulation (repression) | Typically NGG* | - Lacks nuclease activity, allowing modulation of gene expression without DNA cleavage
- Cannot produce permanent edits (e.g., knockouts or knockins)
|
| Cas12a | Knockin & multiplexed genome editing | TTTN | - Generates staggered DSBs
- Able to process its own CRISPR RNA (crRNA), which can simplify multiplexed genome editing
|
*For Cas9 nickase, prime editors, dCas9, and base editors, the PAM is generally derived from the underlying SpCas9 system (NGG).
Depending on the application and the research question, CRISPR-Cas9 systems can be used in a reverse genetics approach, where the function of a gene is studied or modulated. Alternatively, CRISPR-Cas9 systems can be used to generate libraries that serve as powerful forward genetics tools for large-scale functional genomic screens. By designing gRNAs targeting each gene in the genome or specific sets of genes, screens can be conducted to identify genes associated with phenotypes, pathways, or diseases.
Practical tips for effective CRISPR vector design*
Optimizing CRISPR experiments- particularly with respect to selecting the right Cas9 variant, target region, gRNA, and delivery method- is crucial for achieving successful and reproducible results. Here are some design tips summarized from our years of gene delivery experience to guide you through your CRISPR vector design process:
1. Cas9 selection: Cas9 selection is a critical decision to ensure alignment between enzyme function and application, and the choice largely depends on experimental goals and the vector system (see table above). VectorBuilder offers a wide range of codon optimized Cas9 variants tailored to specific experimental objectives.
2. Target site selection: The choice of target site for Cas9 activity depends on the experimental objectives and application. For gene knockouts or knockins, protein-coding regions (exons) are typically chosen as target sites. For gene regulation studies, non-coding regulatory regions (promoters, enhancers, introns, etc.) are targeted.
3. gRNA selection, single vs dual gRNAs: In most cases, e.g. for simple gene knockouts, a single gRNA paired with the right Cas9 generates the desired phenotype. Dual gRNAs are a better choice in specific cases: (1) when using Cas9(D10A) nickase to target opposite strands of a single site, reducing off-target effects as it requires both gRNAs to generate DSBs, (2) to delete a fragment of DNA between two DSBs targeted by a gRNA pair, or (3) to target two different genes simultaneously.
4. PAM and gRNA compatibility: gRNAs must be designed with high on-target efficiency and specificity but low chances of off-target effects. Importantly, the gRNA must target a region within close proximity of the PAM sequence, which depends on the selected Cas9 variant. Our
online vector design studio streamlines the process by designing compatible gRNAs and Cas9 with the correct PAM sequences. Additionally, you can find compatible PAM sequences for various Cas9 variants
here.
5. CRISPR vector selection, all-in-one vs separate vectors: For the CRISPR system to successfully edit the genome, target cells must co-express Cas9 and gRNA simultaneously, either both from a single vector or using separate vectors. All-in-one vectors simplify the process by delivering all necessary components in a single step, ensuring efficient co-expression. In contrast, separate vectors require co-transduction, which can be challenging as some cells may only receive one vector. However, using separate vectors can be advantageous when using larger components, for instance modified Cas9 proteins with tags or markers. Using separate vectors can be especially desirable when cells or organisms are transduced for
stable expression of Cas9 (continuous or
inducible), providing flexibility for subsequent delivery of gRNA sequences, though this method is time and labor intensive.
6. Reporter system selection: Incorporating
selectable markers (e.g., antibiotic resistance) or
fluorescent reporters (e.g., GFP, RFP) into your CRISPR vectors helps with identification and isolation of successfully transduced/edited cells. This step can significantly enhance experimental efficiency and accuracy. Our CRISPR vectors can accommodate several markers, reporters, and tags. Find out more about selecting the right fluorescent marker for your experiment
here.
7. Delivery system selection: Delivery methods vary based on target cells' identity and location as well as the application type. Viral vectors (e.g., lentivirus or AAV) can provide high transduction efficiency and options for transient or long-term expression, making them ideal for hard to transfect cells and in vivo applications. However, they are more complex to produce and can cause higher immunogenicity and toxicity. Non-viral methods, like lipid nanoparticle based transfection or electroporation, are simpler to implement but are typically associated with lower efficiency and can be challenging to deliver to certain cell/tissue types. Find detailed information about various CRISPR delivery methods
here.
8. HDR system selection: For HDR-based precise editing, ssODNs are typically used for inserting short sequences (<60 bp), like small protein tags or point mutations, while dsDNA donors enable the knockin of larger sequences (up to 4-5 kb), such as fluorescent tags or reporter genes. VectorBuilder provides custom donor DNA templates to facilitate precise
HDR repair.
*Note that the design tips provided above are for Cas9 and its variants and not Cas12a.
The success of a CRISPR experiment depends on several factors including careful selection of the appropriate Cas9 variant, gRNA, and delivery system. Many of the considerations discussed here are incorporated into the various systems in the Vector Design Studio, and our design team is always on hand to help with all your CRISPR needs.
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