Table 5.

Troubleshooting advice

Step	Problem	Potential Causes	Solutions
3A(xix)/3B(x)	epegRNA cloning fails: No colonies observed after cloning	For 3A(xix): The presence of many red colonies indicates backbone bleedthrough due to the incomplete digestion of the pU6-tevopreq1-GG-acceptor plasmid.	Repeat digestion with BsaI, PvuII, and NcoI and perform subsequent gel extraction
		For 3A(xix): Oligos not properly phosphorylated	Check that PNK is being performed correctly if sgRNA scaffold oligos (Golden Gate part 2) were not purchased with 5′ phosphorylation
		Overhangs incorrectly designed	Check overhang design, switch between 3A and 3B to try different methods
		Incorrect antibiotic used	All epegRNA and pegRNA plasmids based on our designs yield carb/amp resistance
7	PCR amplification of mRNA transcription template plasmid fails	Non-specific amplification	Re-optimize PCR conditions to avoid aberrant primer binding: rerun the PCR(s) with different annealing temperatures and extension times. Ensure in vitro transcription forward and reverse primers are PAGE purified. Use gel electrophoresis to verify product purity.
		Low-yield amplification	Scale up PCR beyond the suggested volume; then pool and concentrate products in a single silica column. Use gel electrophoresis to verify product purity.
18	mRNA gel electrophoresis shows wrong length transcript, smear, or no transcript	Incorrect length indicates sub-optimal input DNA quality	Check gel electrophoresis from Step 7. A high-quality DNA amplicon input is important. Refer to Step 7 troubleshooting.
		Smear on mRNA gel indicates RNAse contamination	Determine the source of any RNAse contamination. Repeat mRNA prep from Step 8 with RNAse-free technique.
		No Transcript: Sub-optimal input DNA quantity; precipitated RNA pellet lost during LiCl cleanup (Steps 12–17)	Ensure that IVT reaction is initiated with 1 μg of template DNA. Take care during the ethanol washes of the LiCl precipitation to not remove the pellet from the spin tube. Review IVT setup to ensure all reagents are included and in good condition. Ensure that technique is RNAse-free.
41–46	Observed editing rates are low or undetectable in workhorse cell line (HEK293T, N2A, etc.)	PBS and RTT are not optimized	Try more PBS and RTT lengths and combinations.
		Inefficiently edited protospacer	Confirm Cas9 nuclease or base editing activity at that protospacer. Test more protospacers.
		Lipid has oxidized and prevented efficient transfection	Repeat with fresh lipid and Opti-MEM.
		Poor quality plasmids	Re-prep plasmid: run plasmid on a gel to ensure no RNA contamination, which manifests as a low-MW smear on EtBr gel.
		Editor not being delivered	Use a western blot to test for editor expression; transfect easily monitored plasmid such as pmaxGFP to ensure transfection is working.
		SNP in spacer relative to consensus HG38 sequence or other reference sequence	Sequence unedited cells from sample to check for this, adjust epegRNA components accordingly.
		Not using optimal PE systems	Switch to epegRNA, use max architecture, or try PE4 or PE5.
		epegRNA was incorrectly designed: edit not encoded in the 3′ epegRNA extension (causing the RT to synthesize the WT sequence), or the mutation was included in the spacer, preventing Cas9 from binding to the target locus	Check epegRNA design; use one of several web tools to re-design epegRNA and compare output with your epegRNA.
		5′ G not included, transcription from U6 promoter is inefficient (if using U6 promoter for epegRNA transcription)	Ensure that either the spacer sequence begins with a 5′ G, or if it does not, append an extra G at the 5′ end to extend the spacer length to 21 nt.
		First nucleotide in epegRNA 3′ extension is a cytosine	We have observed that 3′ extensions starting with a cytosine generally result in lower prime editing. Redesign epegRNA RTT lengths to avoid starting the 3′ extension with a cytosine.
		epegRNA contains a polyU stretch, which causes premature transcriptional termination of epegRNA	If the polyU stretch is in the RTT, consider adding a silent edit (if possible) to disrupt the polyU sequence. If the polyU stretch is in the spacer, consider targeting an alternative protospacer.
		General technical issues	To parse apart epegRNA problems from experimental workflow errors, check that you can perform high-efficiency prime editing at a previously established site and with a validated edit.
41–46	Efficient editing in workhorse cell line, but inefficient editing in other cell types	Prime editor may not be expressing	Check editor expression with nuclease-mediated indel activity, base editor activity, or western blot. Re-optimize transfection or electroporation protocol, or change from plasmid to mRNA delivery.
		Disconnect between cell line used for optimization and cell line of interest	Re-optimize nicking sgRNA in target cell line. Consider using epegRNAs and/or MLH1dn if these were excluded from initial optimizations, as these modifications tend to have a large impact in more challenging cell lines.
30	PCR1 amplification fails	Cell lysis is incomplete	If using complete mammalian cell lysis buffer (See Reagent Setup), confirm lysis buffer is pH 8.
		PCR1 conditions may not be optimal	Try new combinations of PCR1 primers. Re-optimize thermal cycling steps, in particular the annealing temperature. Repeat the PCR1 with different gDNA template inputs, but keep gDNA input into each PCR consistent across reactions and make sure an adequate number of cells are analyzed. Run control PCRs of previously validated PCR1 primer sets (i.e., HEK3) to confirm that the lysis step worked properly. Use NCBI’s Primer-BLAST to verify that primer pairs do not bind undesired regions.
41– 46	High rates of indel incorporation	Nicking sgRNA is not optimal	Test more nicking sgRNAs, especially PE3b/PE5b nicking sgRNAs if possible.
		MMR is inducing high indels	Switch to PE4 or PE5 system.