Design Tips for Prime Editing

By Emily P. Bentley

Emily P. Bentley January 23, 2025

We recently updated our blog post on Prime Editing, and that meant rereading many of the original papers reporting various prime editing tools. These papers are chock full of great tips to guide your experimental design, especially the design of the RNA sequences you’ll use in prime editing. We thought we’d collect those tips for you in one place!

First, a quick refresher: prime editing is a “search and replace” genome editing technique that relies on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The pegRNA consists of a CRISPR gRNA extended to include a primer binding site (PBS) complementary to the genome and a reverse transcriptase (RT) template including the desired edit.

CRISPR prime editing schematic. The parts of the prime editor and pegRNA are indicated. The prime editor consists of a Cas9n H840A nickase fused to a reverse transcriptase (RT), while the pegRNA includes spacer, scaffold, RT template, and primer binding site (PBS) sequences. The desired edit is part of the RT template. In the first step, these components form a complex, bind target DNA, and nick the Cas9 non-target strand. Next, the primer sequence binds the freed non-target strand, with the PBS hybridizing with genomic DNA. The RT extends the nicked strand using the RT template, incorporating the edit into target DNA. Finally, the DNA is freed from the prime editing complex, and cellular endonucleases and mismatch repair resolve the heteroduplex.

Figure 1: Prime Editing schematic. Created with BioRender.com.

The pegRNA specifies both the genomic location to be edited and the sequence of the edit, so its design changes for every new prime editing application. And optimizing its sequence is important, as prime editing efficiency is affected by the type of edit, genomic context, delivery strategy, cell type, and goal of the experiment.

Now, onto the tips!

General principles

pegRNA design

If you’re just starting out with prime editing, you’ll need to design and optimize a pegRNA for your particular edit. These tips are a great starting point:

Test different lengths of the primer binding site, starting with a length of about 13 nt (Anzalone et al., 2019).
Primer binding sites with 40–60% G/C content are most likely to be successful, although sequences with G/C content outside this range can still be optimized (Anzalone et al., 2019).
Test different lengths of reverse transcriptase template, starting with about 10–16 nucleotides. For longer templates, it is even more important to test different lengths and sequences, as unintended secondary structures in your pegRNA could inhibit editing (Anzalone et al., 2019).

A cartoon of a prime editor bound to a pegRNA, indicating suggested starting lengths for optimization. The primer binding site (PBS) is shown to be about 13 nucleotides long, while the reverse transcriptase template (RTT) is 10 to 16 nucleotides long.

Figure 2: Starting lengths for optimizing your pegRNA primer binding site (PBS) and reverse transcriptase template (RTT). Created with BioRender.com.

The first base of the 3′ extension of the pegRNA should not be C. A C base is speculated to base pair with G81 of the gRNA (Anzalone et al., 2019), disrupting its canonical structure and Cas9 binding (Nishimasu et al., 2014).

A cartoon comparing pegRNA sequences. A pegRNA whose 3′ extension starts with a C base is shown with the C forming an incorrect base pair with G81 of the pegRNA, which causes misfolding of the gRNA scaffold and no prime editor binding. A pegRNA without a C base in this position is shown with canonical gRNA structure bound to the prime editor.

Figure 3: pegRNAs whose 3′ extension begins with a C base can cause unwanted base pairing (left), while pegRNAs that use other bases in this location bind to Cas9 with canonical sgRNA structure (right). Created with BioRender.com.

What to edit

The sequence you choose to install can influence the success of your prime edit. If possible, consider following these suggestions:

Edit the PAM along with your intended edit. This prevents the Cas9 nickase from re-binding and nicking the newly synthesized strand before heteroduplex resolution, which can lead to indels (Anzalone et al., 2019).

A cartoon of a prime editor with two different edit sequences. The DNA sequences are shown with one strand edited and a 5′ DNA flap, before heteroduplex resolution and DNA repair. The first edit has an unchanged PAM. This DNA is shown connected to the prime editor by a two-way arrow, indicating that the editor can re-bind. Re-nicking is represented by scissors and would remove the newly edited DNA. The second edit has an altered PAM. A one-way arrow leads from the prime editor to this edit, indicating that the changed PAM prevents the editor from re-binding.

Figure 4: Editing the PAM prevents the prime editor from re-engaging with DNA it has already edited. Created with BioRender.com.

Add silent mutations near point mutations to create 3-base (or longer) tracts of edited bases. DNA mismatch repair (MMR) is less efficient at identifying these “bubbles” of mismatched bases, enhancing correct editing efficiencies (Chen et al., 2021).

A cartoon depicting mismatch repair (MMR) as a monster with sharp teeth. The monster looks angrily at a DNA sequence with a single base mismatch, but it is confused by a DNA sequence with a 3-base mismatch “bubble.”

Figure 5: MMR efficiently targets single-base mismatches (left), but “bubbles” of 3 or more mismatched bases can often evade MMR (right). Created with BioRender.com.

For specific prime editing tools

There are a lot of different prime editing tools to choose from, and many have tool-specific best practices. Here are some tips for optimizing your tool of choice.

Nicking sgRNAs

PE3 and PE5: These tools use an sgRNA to guide nicking of the unedited strand and encourage the cell to use the edited strand as a template during DNA repair. Test multiple nick sites, starting with sites about 50 bp upstream and downstream from the prime editing nick, and monitor indel frequencies (Anzalone et al., 2019).

A cartoon of two prime editors shown binding to DNA in opposite orientations at sites 50 bases apart. One editor is using a full-length pegRNA as a guide and is bound to DNA at the pegRNA target site. The other prime editor is using an sgRNA as a guide, lacking the 3′ extension including the PBS and RTT, and is bound to DNA in the opposite orientation at a potential sgRNA nick site. Because of the opposite orientations of the two editors, the scissor symbol on each is shown nicking opposite strands of DNA.

Figure 6: Prime editors bound to pegRNAs drive editing (left), while prime editors bound to sgRNAs only drive nicking (right). Start with nicking sgRNAs ~50 bp from the prime editing nick. Created with BioRender.com.

PE3b and PE5b: In this approach, the nicking sgRNA is designed to bind only after the edit is installed. This reduces concurrent nicks, which lowers indel rates (Anzalone et al., 2019). When possible, the PE3b/PE5b approach is recommended over the PE3/PE5 approach.

A cartoon of the PE3b approach to designing nicking sgRNAs. First, a prime editor installs an edit on one strand of DNA. Then, an sgRNA guides a prime editor to nick the opposite strand with a guide sequence complementary to the newly edited sequence, ensuring non-edited DNA cannot be nicked.

Figure 7: Nicking sgRNAs can be designed to target edited DNA, so that nicking only occurs after the edit is installed on the opposite strand. Created with BioRender.com.

MMR inhibitors

PE4 and PE5: These approaches inhibit MMR to improve editing efficiency, but this also allows certain unintended edits to escape cellular surveillance. To limit unintended incorporation of the pegRNA scaffold sequence into the genome, ensure it is not homologous to the target genomic sequence (Chen et al., 2021).

A cartoon comparing two editing outcomes in two different cellular contexts. The top row shows a correct prime edit, while the bottom row shows a prime editor where the reverse transcriptase has over-extended genomic DNA by using the pegRNA scaffold sequence as a template. This outcome is more likely when the pegRNA scaffold is homologous to the target genomic sequence. Both these editing outcomes are shown first in a cellular context with normal mismatch repair (MMR). MMR, depicted as a monster, targets both edit results, hindering correct editing, but also preventing insertions of the pegRNA scaffold sequence into the genome. A large red X is shown over the DNA with an incorrect scaffold insertion. Next, both editing outcomes are shown in a cellular context where MMR is inhibited. The MMR monster is shown very faintly, indicating its absence. In this context, both correct edits and pegRNA scaffold insertions are enhanced. As an alternative strategy to prevent scaffold insertions, the figure recommends avoiding homology between the pegRNA scaffold and the genomic target.

Figure 8: Homology between the pegRNA scaffold sequence (orange) and the genomic target can increase the odds of incorrect editing outcomes, especially when MMR is inhibited. Created with BioRender.com.

RNA protectors

epegRNAs: epegRNAs include structured motifs at their 3′ end to protect them from degradation. When designing epegRNAs with large structured motifs like mpknot, use the pegRNA Linker Identification Tool (pegLIT) to create linkers that minimize unwanted intra-RNA base pairing with the primer binding site (Nelson et al., 2022).
PE7: This editor includes the fused RNA-binding protein La to protect the end of the pegRNA. Adding 3′ polyU tracts to the end of pegRNAs (but not epegRNAs) can improve either endogenous or fused La binding (Yan et al., 2024).

A cartoon depicting two different strategies for protecting the 3′ ends of pegRNAs. epegRNAs include a 3′ highly structured RNA pseudoknot. PE7 includes the RNA-binding protein La fused to the prime editor. The pegRNA is shown with an extended 3′ polyU tail bound by PE7.

Figure 9: epegRNAs (left) and PE7 (right) offer two different strategies for protecting pegRNAs from degradation. Created with BioRender.com.

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That’s all our tips for today! Each tip includes a citation to the paper it was drawn from. If we’ve piqued your curiosity, we encourage you to check out the original papers for more details.

You can find many software programs to help you design pegRNAs. Here on the Addgene blog, we featured a guest blog by the developer of PRIDICT, which helps users select pegRNA designs that are most likely to drive efficient editing. This is not the only available software, however. You may find it useful to ask a mentor what software tools they recommend — feel free to share your favorite software in the comments below!

References and Resources

References

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

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

Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001

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

Additional Resources on the Addgene blog

Resources on Addgene.org

Topics: CRISPR, Molecular Biology Protocols and Tips, CRISPR Protocols and Tips, Other CRISPR Tools

Design Tips for Prime Editing