Prime Editing: Adding Precision and Flexibility to CRISPR Editing

Prime Editing: Precise and Flexible CRISPR Editing

Over 75,000 pathogenic genetic variants have been identified in humans and cataloged in the ClinVar database. Previously developed genome editing methods using nucleases and base editors have the potential to correct only a minority of those variants in most cell types. But prime editing, a CRISPR technique developed in David Liu’s lab in 2019, has added considerably more precision and flexibility to the CRISPR editing world.

The approach, originally published in Nature, is a “search-and-replace” genome editing technique that mediates targeted insertions, deletions, and all possible base-to-base conversions (Anzalone et al., 2019). Plus, it can combine different types of edits with one another. All of this is possible without double-strand breaks (DSBs) or donor DNA templates. 

Figure 1: An overview of prime editing. Created with BioRender.com.

How does it work? First, an engineered prime editing guide RNA (pegRNA) that both specifies the target site and contains the desired edit (or edits) engages the prime editor protein. The prime editor consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase is guided to the DNA target site by the pegRNA — a guide RNA that also encodes the desired edit and homology to the genomic DNA locus. After nicking by Cas9, the homologous pegRNA sequence hybridizes to the target site, and the reverse transcriptase domain copies the desired edit, directly polymerizing new DNA onto the nicked target strand.

The initial result is a heteroduplex, with overlapping strands of edited and unedited DNA. The heteroduplex is resolved by the cell’s mismatch repair system, and more recent prime editing innovations have improved the odds of mismatch repair favoring the edited strand.

The first iteration of prime editor simply fused the wild-type Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase to the C-terminus of Cas9 H840A nickase (PE1). But innovations by the Liu lab and others have led to a variety of prime editing tools. We’ll summarize the major ones in Addgene’s repository later in the post.

Advantages of prime editing

Less constrained by PAM sequence location

The prime editor extends the reach of CRISPR genome editing, as it can edit near to or far from PAM sites, making it less constrained by PAM availability than other methods. The PAM-to-edit distance can be over 30 base pairs for prime editing. Since PAM sites occur on average every ~8 base pairs on either DNA strand, many previously developed base editors with a <8 base-pair editing window cannot edit within what Fyodor Urnov refers to as “PAM deserts” in the genome.

More versatile and precise than base editing (in certain circumstances)

Current base editors can perform any base-to-base conversion in principle, but some changes require multiple steps and enzymes. Prime editing can encode any of the 12 possible base-to-base changes in a single edit.

Prime editing is also more precise. Base editors, for example, typically edit all the target bases within the base editing window, while prime editors make a specific edit defined by the pegRNA. In cases when bystander editing is unacceptable, prime editors can be used to avoid this possibility.

However, there are instances where traditional base editors are preferred. For instance, if target nucleotides are positioned within the canonical base editing window, base editing has higher efficiency and fewer indels than prime editing. But for positions that aren’t well positioned within the editing window, prime editing is more efficient due to its lower dependence on PAM placement.

Fewer byproducts and more efficient than homology-directed repair

Homology-directed repair (HDR) stimulated by double strand breaks has been widely used to generate precise changes. However, the efficiency of Cas9 cleavage is relatively high while the efficiency of HDR is relatively low, meaning that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result, Cas9 treatment causes most products to be indels while the efficiency of HDR is typically less than 10%.

In contrast, the original paper introducing prime editing demonstrated ~20-50% efficiency in HEK293T cells with 1-10% indels. Since then, further innovations have continued to improve the maximum efficiency of prime editing. While maximum efficiency isn’t everything — prime editing typically needs to be optimized for each application, and the efficacy varies widely — it still almost always results in higher ratios of desired edits to indel byproducts than Cas9-initiated HDR.

Prime editing tools

Prime editing is precise and versatile, but it requires careful optimization to achieve the best efficiency. The type of edit, genomic context, delivery strategy, cell type, and goal of the experiment can all impose different constraints and affect how you select and design your prime editing tools.

Here, we’ll briefly cover some major advances in prime editing tools. Scroll down to see a table summarizing all of them!

PE2: Improving reverse transcriptase efficiency

Building upon prior reverse transcriptase research, the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, enhance binding between the template and primer binding site, increase processivity, or improve thermostability. What came out on top? The Cas9 nickase fused to a pentamutant of M-MLV RT. They called this system PE2, which had prime editing efficiencies on average 2.3- to 5.1-fold (though up to 45-fold) higher across different genomic sites compared to PE1 (Anzalone et al., 2019).

PE3: Resolving mismatched DNA to favor the edit

Once the prime editor incorporates the edit into one strand, there’s a mismatch between the original sequence on one strand and the edited sequence on the other strand. To guide heteroduplex resolution to favor the edit, the Liu lab turned to a strategy they previously used when they developed base editing. By nicking the non-edited strand, they can cause the cell to remake that strand using the edited strand as the template.

The PE3 system does just this by including an additional sgRNA. Using this sgRNA, the prime editor nicks the unedited strand away from the initial nick site (to avoid creating a double strand break), increasing editing efficiencies 2-3-fold. However, the process of double nicking increases indel formation slightly. Designing the sgRNA with a spacer that only binds the edited strand, as in the PE3b system, guides nicking of the unedited strand only after editing has occurred and reduces indels by 13-fold (Anzalone et al., 2019).

PE3, as originally introduced, relies on the PE2 enzyme.

PE4 and PE5: Engineering mismatch repair

Even in PE3 and PE3b, prime editing efficiency can vary widely — so its inventors kept optimizing it. Using a pooled CRISPRi screen, the Liu lab discovered that cellular mismatch repair contributed to a large number of unintended prime editing outcomes, including indels. Therefore, they engineered a dominant-negative mutant of the protein MLH1, a component of the MutSα–MutLα mismatch repair (MMR) complex, to temporarily inhibit this process and promote copying of the edited strand to the unedited strand (Chen et al., 2021). The team applied this approach to both the PE2 and PE3 systems, naming the new systems PE4 and PE5, respectively. These systems improved editing efficiency by 7.7-fold (PE4 versus PE2) and 2.0-fold (PE5 versus PE3).

How exactly does inhibiting DNA repair allow for correct heteroduplex resolution? The prime editing heteroduplex is likely recognized as an MMR intermediate, and the MutSα–MutLα complex may either selectively excise the edited DNA flap or prevent it from hybridizing with the unedited strand. Temporarily inhibiting this complex may allow time for 5′ flap exonucleases and DNA ligases to act first.

PE4 and PE5, as originally introduced, rely on the PE2 enzyme.

PEmax: Optimizing prime editor sequence to improve expression and activity

In addition to the numerous innovations that streamline the process of prime editing, the Liu lab contributed an improved prime editing “architecture” that can be combined with any of the PE2-PE5 strategies. PEmax contains a reverse transcriptase with a sequence optimized for human codons; additional nuclear localization sequences; and two mutations in Cas9 previously shown to improve nuclease activity (Chen et al., 2021). In a system as complex as prime editing, every efficiency counts!

The optimized enzyme can be used with any of the PE2-5 approaches, sometimes referred to as PE2max, PE3max, etc.

PE6: Specialized prime editing variants

Four years after their initial publication, the Liu lab returned to the reverse transcriptase domain for optimization. They developed phage-assisted evolution strategies to evolve more effective prime editor proteins, but found that the mutations that emerged depended strongly on the target edit (Doman et al., 2023). Therefore, they decided to evolve a range of specialized prime editors (PEs).

PE6a and PE6b are small prime editors with RT domains derived from the E. coli Ec48 retron RT and the S. pombe Tf1 retrotransposon RT, respectively. Their small size comes at the cost of improved efficiency, although both enzymes still approach or exceed PEmax editing efficiencies for short, simple edits.

PE6c and PE6d further evolved the Tf1 and M-MLV RT enzymes, respectively, to obtain PEs small enough to deliver with AAVs but still efficient at long and complex edits.

The team also developed PE6e-g editors with mutations to the Cas9 domain but found that these mutations only improved efficiency for some edits in unpredictable ways. In some cases, however, combining an evolved RT domain from PE6a-d with an evolved Cas9 domain from PE6e-g produced additive improvements. These variants are worth testing if you want to make your prime editing as efficient as possible.

Figure 2: Decision tree for selecting a PE6 variant. PE = prime editor; RTT = reverse transcriptase template; PBS = primer binding site. Image from Doman et al., 2023, under a CC BY 4.0 license.

epegRNAs: Structured RNAs with improved stability

Traditional sgRNAs are protected from cellular degradation by Cas9 binding, but pegRNAs have an extended 3′ tail containing the reverse transcriptase template and primer binding site. If this tail is degraded, the truncated pegRNA can still guide Cas9 and compete with full pegRNAs for access to the target site, but it cannot drive prime editing. To protect the 3′ end, the Liu lab appended RNA pseudoknots, calling this longer RNA an “engineered pegRNA” or epegRNA. Although the addition of the RNA pseudoknot sometimes altered epegRNA expression compared to pegRNAs, prime editing efficiency was improved even in contexts where expression was reduced (Nelson et al., 2022).

PE7: An alternate approach to RNA stability

This recent contribution by the Adamson lab also addresses the problem of RNA stability. Using a CRISPRi screen, they identified a protein that enhances prime editing in mammalian cells: the small RNA-binding exonuclease protection factor La. They found that La, which is ubiquitously expressed in eukaryotes, binds and stabilizes the 3′ tail of pegRNAs (Yan et al., 2024). The team fused La to the C-terminal end of PEmax to produce PE7.

PE7 significantly improves editing efficiencies compared to PEmax with normal pegRNAs, but combining PE7 with epegRNAs resulted in similar or reduced editing efficiencies, likely because both innovations aim to solve the same problem. Both approaches are effective, but users should pick one or the other.

Which prime editor is which again?

We’re glad you asked. Hopefully this table helps you keep them straight!

Innovation	Purpose	PE2	PE3	PE4	PE5	PEmax	PE6	PE7
Pentamutant reverse transcriptase	Higher efficiency	✓	✓	✓	✓	✓		✓
Second sgRNA to guide nicking of the unedited strand	Favor the edited strand during mismatch repair		✓		✓	~	~	~
Transiently co-express mismatch repair inhibitor MLH1dn	Promote desired resolution of heteroduplex			✓	✓	~	~	~
Human codon optimization	Improve prime editor expression	~	~	~	~	✓		✓
SV40 and c-Myc nuclear localization sequences	Improve translocation to the nucleus	~	~	~	~	✓	✓	✓
R221K N394K Cas9 mutations	Improve Cas9 nuclease activity	~	~	~	~	✓	✓	✓
Evolved reverse transcriptase domains	Smaller size and/or improved efficiency						~
Evolved Cas9 domains	Improve efficiency only for certain edits						~
3′ RNA pseudoknot (epegRNA)	Improve pegRNA stability	~	~	~	~	~	~	✖
Fuse RNA-binding protein La to the prime editor	Improve RNA stability							✓

✓ reflects an innovation always included in the indicated tool.
~ reflects an innovation that may optionally be combined with the indicated tool.
✖ reflects an innovation that should not be combined with the indicated tool.

What’s next for prime editing?

In addition to the smorgasbord of prime editing tools discussed here, many additional strategies have been developed to augment prime editing. You can find a brief discussion of some of these strategies in our CRISPR guide. Some examples include:

• TwinPE: two prime editors make complementary edits to opposite strands of the DNA and skip the cellular mismatch repair step (Anzalone et al., 2022)
• PASSIGE and PASTE: two strategies that combine prime editing with a recombinase to install large sequences (up to 10 kb) into the genome (Pandey et al., 2024; Yarnall et al., 2023)

Prime editing has a lot of promise for treating genetic diseases. In our summer Hot Plasmids post, we covered the Liu lab’s first demonstration of prime editing used to rescue a genetic disease phenotype in an animal model, which was made possible through their use of engineered virus-like particles. However, there’s still a long way to go before we see prime editors in the clinic. We look forward to following new advances in this evolving field!

Find prime editing plasmids!

Find plasmids from David Liu's lab!

This post was originally written by Jennifer Tsang in October 2019 and updated by Emily P. Bentley in November 2024.

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References and Resources

References

Anzalone, A. V., Gao, X. D., Podracky, C. J., Nelson, A. T., Koblan, L. W., Raguram, A., Levy, J. M., Mercer, J. A. M., & Liu, D. R. (2022). Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nature Biotechnology, 40(5), 731–740. https://doi.org/10.1038/s41587-021-01133-w

Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149–157. https://doi.org/10.1038/s41586-019-1711-4

Chen, P. J., Hussmann, J. A., Yan, J., Knipping, F., Ravisankar, P., Chen, P.-F., Chen, C., Nelson, J. W., Newby, G. A., Sahin, M., Osborn, M. J., Weissman, J. S., Adamson, B., & Liu, D. R. (2021). Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22), 5635-5652.e29. https://doi.org/10.1016/j.cell.2021.09.018

Choi, J., Chen, W., Suiter, C. C., Lee, C., Chardon, F. M., Yang, W., Leith, A., Daza, R. M., Martin, B., & Shendure, J. (2022). Precise genomic deletions using paired prime editing. Nature Biotechnology, 40(2), 218–226. https://doi.org/10.1038/s41587-021-01025-z

Doman, J. L., Pandey, S., Neugebauer, M. E., An, M., Davis, J. R., Randolph, P. B., McElroy, A., Gao, X. D., Raguram, A., Richter, M. F., Everette, K. A., Banskota, S., Tian, K., Tao, Y. A., Tolar, J., Osborn, M. J., & Liu, D. R. (2023). Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell, 186(18), 3983-4002.e26. https://doi.org/10.1016/j.cell.2023.07.039

Lin, Q., Jin, S., Zong, Y., Yu, H., Zhu, Z., Liu, G., Kou, L., Wang, Y., Qiu, J.-L., Li, J., & Gao, C. (2021). High-efficiency prime editing with optimized, paired pegRNAs in plants. Nature Biotechnology, 39(8), 923–927. https://doi.org/10.1038/s41587-021-00868-w

Nelson, J. W., Randolph, P. B., Shen, S. P., Everette, K. A., Chen, P. J., Anzalone, A. V., An, M., Newby, G. A., Chen, J. C., Hsu, A., & Liu, D. R. (2022). Engineered pegRNAs improve prime editing efficiency. Nature Biotechnology, 40(3), 402–410. https://doi.org/10.1038/s41587-021-01039-7

Pandey, S., Gao, X. D., Krasnow, N. A., McElroy, A., Tao, Y. A., Duby, J. E., Steinbeck, B. J., McCreary, J., Pierce, S. E., Tolar, J., Meissner, T. B., Chaikof, E. L., Osborn, M. J., & Liu, D. R. (2024). Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-024-01227-1

Yan, J., Oyler-Castrillo, P., Ravisankar, P., Ward, C. C., Levesque, S., Jing, Y., Simpson, D., Zhao, A., Li, H., Yan, W., Goudy, L., Schmidt, R., Solley, S. C., Gilbert, L. A., Chan, M. M., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson, B. (2024). Improving prime editing with an endogenous small RNA-binding protein. Nature, 628(8008), 639–647. https://doi.org/10.1038/s41586-024-07259-6

Yarnall, M. T. N., Ioannidi, E. I., Schmitt-Ulms, C., Krajeski, R. N., Lim, J., Villiger, L., Zhou, W., Jiang, K., Garushyants, S. K., Roberts, N., Zhang, L., Vakulskas, C. A., Walker, J. A., Kadina, A. P., Zepeda, A. E., Holden, K., Ma, H., Xie, J., Gao, G., … Gootenberg, J. S. (2023). Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nature Biotechnology, 41(4), 500–512. https://doi.org/10.1038/s41587-022-01527-4

Additional resources on the Addgene blog

• PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs
• Visit our CRISPR topic page
• Learn the basics of CRISPR
• Download the CRISPR 101 eBook

Resources on Addgene.org

• Read the CRISPR guide
• Find CRISPR plasmids by function
• Browse our entire plasmid collection