Comparison of CRISPR-Cas9, base editing, and prime editing
| Key features | CRISPR-Cas9 | Base editing | Prime editing |
|---|---|---|---|
| Mechanism of action | Utilizes a Cas9 nuclease and a gRNA to create DSBs at specific DNA loci. | Uses a Cas9 nickase fused to a deaminase enzyme to convert one nucleobase to another without introducing DSBs. | Involves a Cas9 nickase fused to a reverse transcriptase domain and uses pegRNAs to install insertions, deletions, or substitutions. |
| Types of edit | Disrupts or replaces targeted DNA via NHEJ or HDR. | Enables precise single-nucleotide modifications, such as C-to-T or A-to-G at specific locations. | Capable of making more varied edits, such as base changes and small insertions/deletions, without generating full DSBs. |
| DNA breaks required | Yes, it creates DSBs. | Typically, only a nick is made in one strand. | No DBS breaks. Instead, it relies on a nick in a single strand for polymerase extension. |
| Off-target considerations | Off-target cleavage can occur if the gRNA has partial homology to non-target sites, but high-fidelity variants reduce this risk. | Generally, there are fewer off-target edits compared to Cas9-induced DSBs, but some off-target deaminations may still occur. | This method generally has reduced off-target activity compared to standard CRISPR-Cas9, although optimization is ongoing. |
| Efficiency and accuracy | While often highly efficient, results can vary depending on the cell type and repair pathway. | The precision for single-base changes is typically high, with efficiency depending on the accessibility of the target site and the surrounding base context. | It offers moderate to high precision, but efficiency can be lower than base editing, with ongoing improvements to pegRNAs and enzymes. |
| Tissue-specific efficiency | Success depends on vector choice, promoter specificity, and cellular repair machinery. | Similar factors affect the efficiency, which may vary depending on the tissue or cell line due to differences in deaminase expression and DNA accessibility. | It is currently under active investigation and optimal tissue tropism depends on delivery methods and pegRNA design. |
| Toxicity profile | Potential for cytotoxicity resulting from DSBs, which can be improved by using high-fidelity Cas9 variants. | This is typically lower cytotoxicity since no DSBs are created, but the risk of off-target deamination remains a concern. | There is a lower risk of large genomic rearrangements, but low-level off-target events are still possible. |
| Stage of development | Widely studied and is being used in multiple clinical trials, as well as in ex vivo therapies. | There is growing clinical interest with some proof-of-concept studies conducted in genetic and acquired diseases. | The technology is rapidly evolving with preclinical studies showing high promise, but broad therapeutic use is still in progress. |
| Complexity and size | This technology requires minimal resources: only a single Cas9 protein plus gRNA (100 nt). | The combination of Cas9 nickase and deaminase adds approximately 1 kb of extra size, requiring careful design to prevent off-target changes. | The system includes Cas9 nickase, reverse transcriptase, and more complex pegRNAs for design flexibility. |
DSB: double-stranded break; gRNA: guide RNA; HDR: homology-directed repair; NHEJ: non-homologous end joining; pegRNA: prime editing gRNA; CRISPR: clustered regularly interspaced short palindromic repeat; Cas: CRISPR-associated