Table 1.

Use cases for various PE systems and modifications

PE system	Uses
PE1 Cas9 (H840A)–WT RT	Not recommended; PE1 was the prototype prime editor from which PE2 was developed.
PE2 Cas9(H840A)–engineered RT	PE2 yields lower editing than PE3-5. However, PE2 may be preferred if: • Secondary nicks from PE3/PE5 generate an unacceptable frequency of indels, and long-term MLH1dn expression in the PE4/PE5 systems is not desired • If the application does not require optimized editing levels (i.e., creating a cell line), PE2 is the simplest and fastest method, as a nicking guide does not need to be optimized • If high editing efficiency is achieved without PE3-5 systems, for example due to the MMR-evading nature of the edit, or the addition of silent nearby mutations
PE3 / PE3b PE2 + Additional nicking sgRNA (PE3b if nicking sgRNA protospacer overlaps with edit)	PE3 and PE4 offer similar editing efficiencies; if PE3 does not generate substantial indels at the target locus and yields high editing efficiency, then it can serve as a good choice. Importantly, the relative editing of PE3 and PE4 varies by cell type. PE3 also provides the highest editing efficiency without inhibiting cellular MMR. Note: Several nicking sgRNAs (positioned both upstream and downstream of the edit) should be screened for optimal editing efficiency and a high editing:indel ratio. If an appropriate PAM exists, PE3b nicking sgRNAs should be screened as well and will usually provide the highest efficiencies and fewest indel byproducts.
PE4 PE2 + MLH1dn	PE4 is most useful when indels at the target site must be minimized or in applications that cannot use nicking sgRNAs; it yields improved editing relative to PE2, but its efficiency relative to PE3 varies depending on cell type. Note: cellular effects of long-term MLH1dn expression (>5 days) have not been assessed. If MLH1dn expression could interfere with downstream experiments, do not use. Note: of less benefit compared to PE2 in MMR-deficient cell types.
PE5 / PE5b PE2 + Additional nicking sgRNA + MLH1dn (PE5b if nicking sgRNA protospacer overlaps with edit)	PE5 typically yields the highest editing efficiency out of all PE systems, and offers substantially reduced indels compared to PE3. Note: cellular effects of long-term MLH1dn expression (>5 days) have not been assessed. If MLH1dn expression could interfere with downstream experiments, do not use. See PE3 information for notes on nicking sgRNA design. Note: of less benefit compared to PE3 in MMR-deficient cell types.
Protein Architecture	Uses
Original architecture15	Not recommended; no longer state-of-the-art.
Max architecture30 (Addgene #174820)	Use for all applications. The max architecture is always the same as or better in editing efficiency than the original architecture across all edits and cell types tested.
pegRNA	Uses
pegRNA	Not recommended unless practical limitations such as chemical synthesis limitations prevent the use of epegRNAs.
epegRNA	Recommended for all applications: epegRNAs almost always offer high editing efficiencies than pegRNAs across all edits and cell types tested.
Silent mutations	Uses
None	If a given application does not allow silent mutations to be incorporated (efficient prime editing can still be achieved without them).
PAM (or seed)-disrupting mutations	Recommended if possible. Disruption of the PAM or seed region reduces re-binding and nicking of the edited product. Note: check a codon usage table to ensure that the mutations are silent and that the silent changes do not create a highly disfavored codon.
MMR-evading mutations	Installing multiple contiguous or tightly clustered mutations can help increase editing efficiency, especially if the PE2 system is being used. Different silent or benign mutations, in addition to the desired edit alone, should be tested whenever possible. Note: check a codon usage table to ensure that the mutations do not use highly disfavored codons.

For a given prime editing experiment, one option from each category above is selected. When selecting PE systems and the incorporation of silent mutations, though, the optimal version will depend on the edit, cell type, and application. For these decisions, empirical testing for each site and mutation is needed to ensure optimal editing.