What is CRISPR base editing?
CRISPR base editing is a genome editing technique that enables the direct, irreversible conversion of one DNA base into another without introducing DSBs or requiring donor DNA templates. This method is particularly useful for correcting point mutations, which account for a significant portion of known genetic diseases.
How it works
Base editors are engineered proteins that combine:
- A Cas9 nickase (nCas9), which makes a single strand cut in DNA
- A deaminase enzyme, which chemically modifies a specific base
There are two main types of base editors:
- Cytosine base editors (CBEs): Convert cytosine (C) to thymine (T), effectively changing a C•G base pair to a T•A pair
- Adenine base editors (ABEs): Convert adenine (A) to guanine (G), changing an A•T base pair to a G•C pair
The guide RNA (gRNA) directs the base editor to a specific DNA sequence, where the deaminase modifies the target base. The cell’s natural repair mechanisms then fix the complementary strand, completing the base conversion.
Advantages
- No double-stranded breaks, reducing the risk of large deletions or chromosomal rearrangements
- High efficiency for single base changes
- Ideal for correcting pathogenic point mutations
What is CRISPR prime editing?
CRISPR prime editing is a more versatile genome editing method that enables the insertion, deletion, or substitution of DNA sequences without requiring double-stranded breaks or donor DNA templates. Developed in 2019, prime editing is often described as a “search-and-replace” tool for the genome.
Prime editors consist of:
- A Cas9 nickase fused to a reverse transcriptase enzyme
- A prime editing guide RNA (pegRNA)that directs the Cas9 to the target site and encodes the desired edit
The pegRNA contains:
- A spacer sequence that guides the Cas9 to the target DNA
- A primer binding site (PBS) and a reverse transcription template that encodes the desired edit
Once the Cas9 nickase creates a single-strand break, the reverse transcriptase uses the pegRNA as a template to insert the new genetic information directly into the genome. This allows for:
- All 12 possible base substitutions
- Small insertions and deletions
- Precise edits without relying on homologous recombination
Prime editing is especially promising for correcting complex mutations that are not addressable by base editing alone. Read more here.
What is the difference between CRISPR base and prime editing?
While both technologies offer precision editing without double-stranded breaks, they differ in scope, complexity, and use cases.
| Feature | Traditional CRISPR-Cas9 | CRISPR base editing | CRISPR prime editing |
| Edit types | Double-strand breaks (DSBs) followed by indels or HDR | Single base transitions (C→T, A→G) | All base substitutions, small insertions/deletions |
| Enzyme components | Cas9 nuclease | Cas9 nickase + deaminase | Cas9 nickase + reverse transcriptase |
| Guide RNA | Standard sgRNA | Standard sgRNA | pegRNA (with RTT and PBS) |
| Double-strand breaks | Yes | No | No |
| Repair mechanism | Non-homologous end joining (NHEJ) or homology-directed repair (HDR) | Direct base conversion via deamination | Reverse transcription and flap resolution |
| Precision | Moderate (risk of indels and off-targets) | High for specific transitions | Very high, broader range |
| Edit scope | Indels, large deletions, HDR-mediated insertions | C→T and A→G transitions | All base changes, small insertions/deletions |
| Complexity | Simple design, but less predictable outcomes | Moderate design, predictable edits | Complex design, highly programmable |
| Efficiency | High in many systems, but variable HDR efficiency | High for compatible targets | Variable, cell-type dependent |
| Limitations | Off-target DSBs, unpredictable indels, low HDR efficiency | Limited transition edits, bystander effects | Complex pegRNA design, lower efficiency in some cell types |
| Feature | Traditional CRISPR-Cas9 | CRISPR base editing | CRISPR prime editing |
| Edit types | Double-strand breaks (DSBs) followed by indels or HDR | Single base transitions (C→T, A→G) | All base substitutions, small insertions/deletions |
| Enzyme components | Cas9 nuclease | Cas9 nickase + deaminase | Cas9 nickase + reverse transcriptase |
| Guide RNA | Standard sgRNA | Standard sgRNA | pegRNA (with RTT and PBS) |
| Double-strand breaks | Yes | No | No |
| Repair mechanism | Non-homologous end joining (NHEJ) or homology-directed repair (HDR) | Direct base conversion via deamination | Reverse transcription and flap resolution |
| Precision | Moderate (risk of indels and off-targets) | High for specific transitions | Very high, broader range |
| Edit scope | Indels, large deletions, HDR-mediated insertions | C→T and A→G transitions | All base changes, small insertions/deletions |
| Complexity | Simple design, but less predictable outcomes | Moderate design, predictable edits | Complex design, highly programmable |
| Efficiency | High in many systems, but variable HDR efficiency | High for compatible targets | Variable, cell-type dependent |
| Limitations | Off-target DSBs, unpredictable indels, low HDR efficiency | Limited transition edits, bystander effects | Complex pegRNA design, lower efficiency in some cell types |
In summary, CRISPR base editing is ideal for making simple, high-efficiency edits to single bases, while CRISPR prime editing offers broader editing capabilities with slightly more complexity in design and delivery.
Applications and limitations
Applications
CRISPR base editing:
- Therapeutic correction of point mutations: Many genetic diseases are caused by single-nucleotide variants (SNVs), making base editing a powerful tool for gene therapy
- Functional genomics: Base editors are widely used in high-throughput screens to study gene function by introducing precise mutations
- Agricultural biotechnology: Base editing can be used to improve crop traits without introducing foreign DNA
CRISPR prime editing:
- Correction of complex mutations: Prime editing can address insertions, deletions, and transversions that base editors cannot
- Disease modeling: Researchers can introduce specific mutations into cell lines or animal models to study disease mechanisms
- Gene therapy: Prime editing holds promise for treating a broader range of genetic disorders, including those involving frameshift mutations or splice site errors
Limitations
CRISPR base editing:
- Limited to specific base transitions (C→T, A→G): They can only correct a subset of point mutations, limiting their utility for edits that require other types of base changes or insertions/deletions
- Risk of bystander edits: Unintended changes to nearby bases within the editing window (typically 4-8 nucleotides upstream of the PAM)
- Editing efficiency can vary: Base editing efficiency is influenced by several factors, including the target sequence context, chromatin accessibility, and the specific base editor used
CRISPR prime editing:
- Lower editing efficiency in some cell types: Primary cells and certain stem cells may exhibit lower editing rates compared to immortalized cell lines
- Complex pegRNA design: Designing effective pegRNAs can be more challenging than standard sgRNAs, requiring careful optimization of length, structure, and sequence context
- Limited editing window: The efficiency of prime editing is highly dependent on the position of the desired edit relative to the nick site
- Potential for unintended edits: Although prime editing reduces off-target effects compared to traditional CRISPR-Cas9, it can still introduce unintended insertions, deletions, or mismatches—especially if the pegRNA is not well-optimized
- Delivery challenges: The relatively large size of the prime editor protein (Cas9 nickase fused to reverse transcriptase) can complicate delivery, particularly for in vivo applications
Designing tools for base and prime editing
Designing gRNAs for CRISPR base editing
In base editing, the guide RNA (gRNA) directs a Cas9 nickase fused to a deaminase enzyme to a specific DNA sequence. The goal is to position the target base within the enzyme’s editing window, typically 4–8 nucleotides upstream of the PAM site.
Key design considerations:
- PAM compatibility: Most base editors use SpCas9, which requires an NGG PAM variants like SpCas9-NG or SaCas9 expand targeting options
- Editing window: Ensure the target base falls within the active editing window of the chosen base editor (i.e., BE4max, ABE8e)
- Bystander bases: Avoid sequences with multiple editable bases in the window to reduce unintended edits
- Strand orientation: Choose the strand that places the editable base in the correct position relative to the PAM
Tools for gRNA Design:
- IDT’s Alt-R™ CRISPR tools offer base editor-specific design modules tat predict editing efficiency and off-target potential.
Designing pegRNAs for CRISPR prime editing
Prime editing uses a more complex RNA molecule called a prime editing guide RNA (pegRNA). This RNA not only guides the Cas9 nickase to the target site but also encodes the desired edit via a reverse transcriptase template.
Components of a pegRNA
- Spacer sequence: Functions like a standard gRNA, guiding Cas9 to the target site
- Primer binding site (PBS): A short sequence (typically 8–15 nt) that anneals to the nicked DNA strand
- Reverse transcriptase template (RTT): Encodes the desired edit and flanking homology
Key design considerations
- Edit positioning: The desired edit should be placed within the RTT so that it is efficiently copied into the genome
- PBS length: Affects binding stability and editing efficiency; often optimized empirically
- RTT length: Typically, 10–30 nt, depending on the size of the intended edit
- Secondary structure: Avoid strong secondary structures in the pegRNA that could hinder reverse transcription
Tools for pegRNA design
- PrimeDesign (Broad Institute) and pegFinder are widely used tools that automate pegRNA design, including RTT and PBS optimization.
- Some platforms also support paired nicking strategies (PE3) to enhance editing efficiency by introducing a second gRNA to nick the non-edited strand.
Synthesis and delivery
Both gRNAs and pegRNAs can be synthesized as:
- Chemically modified RNAs for increased stability and reduced immune response
- In vitro transcribed RNAs for cost-effective, scalable production
- Plasmid or lentiviral constructs for stable expression in cells
Delivery options include:
- Electroporation (especially for RNP or mRNA formats)
- Lipid nanoparticles (LNPs) for in vivo or ex vivo delivery
- Viral vectors (i.e., AAV, lentivirus) for long-term expression
