Table 5.

Troubleshooting.

Step	Problem	Possible reason	Solution
9)	Low number of colonies	Inefficient cloning	Ensure plasmid backbone is high quality. Use fresh enzymes and buffer.
		Incorrect overhangs	Check overhang designs. The cloning can be simulated in Snapgene to confirm that the choice of enzymes and overhang sequences are compatible.
	High frequency of background (red) colonies	Inefficient digestion of plasmid backbone	Ensure plasmid backbone is high quality. Use fresh restriction enzymes and buffer.
41)	Poor transfection efficiency	Cell quality is suboptimal	Use low passage cells (<15). Ensure cell density at time of transfection is 70–80%. Do not let cells in culture reach >80% confluency during maintenance. Ensure cells are seeded evenly by swirling the plate in a figure-of-eight motion after seeding. Re-optimise transfection conditions using a fluorescent plasmid e.g., pMAXGFP
		Plasmid DNA is poor quality	Re-prep plasmids / RNA. Carefully inspect them using a Nanodrop. Do not use if they are low concentration and/or 260/280 is <1.8. Run them on a gel to check for degradation.
		Transfection reagent has gone bad	Replace lipofectamine 3000.
	Poor cell health post-transfection	Transfection is stressful, endotoxin contamination in plasmids	Re-prep plasmids using an endotoxin-free prep kit. Try lowering the overall amount of DNA/RNA transfected per well. Confirm cells were at correct confluency at time of transfection.
60)	PCR amplification fails	No amplification of target band	Ensure primer sequences and concentration are correct. Use the primers for NOLC1 listed in this protocol as a positive control. Ensure cell lysis was complete. Further purify gDNA extracts with magnetic beads. Try different volumes of input gDNA. Re-optimize PCR cycling temperatures, particularly the primer annealing temperature. Try re-designing the round 1 primers.
78)	No measurable editing in workhorse cells (e.g. HEK293FT)	Suboptimal atgRNAs	Use positive control atgRNAs, payload plasmid and NGS primers provided in this protocol. If no editing is observed for positive controls, re-prep plasmids, confirm transfection was successful with pMAXGFP, double check correct use of atgRNAs, payloads and NGS primers. If the components provided in this protocol work but not custom atgRNAs, try different spacers, try more PBS lengths, ensure the 5′ end of the spacer is a G, check design of NGS primers and/or ddPCR primers and probe
		Genomic locus is not permissive to editing	Some genomic loci are just difficult to edit. Try more spacer sequences or switch to a new locus. Consider whether attB and/or payload insertion at this site could be toxic to cells.
		CRISPResso2 parameters are incorrect	Inspect fastq files for reads corresponding to editing. In the terminal, use the command '> grep -c [SEGMENT OF EDITED SEQUENCE] *.fastq' to search and count for edited reads (note that the fastq files must be unzipped and the search string in capital letters). If grep finds edited reads but CRISPResso does not measure them, use the CRISPResso2 documentation to check the parameters used.
	High rate of indels	Suboptimal nicking guide RNA	Test more nicking sgRNAs, switch to twinPE editing
91)	No positive droplets for target amplicon	PCR failure	Use atgRNAs, payload, primers and probes listed in this protocol as a positive control. Test new primer/probe designs. Optimise cycling conditions.
	High target signal in negative control wells	Non-specific amplification from the payload	Try additional restriction enzymes that cut the payload plasmid 5′ of the attP. Re-design forward primer.
	Low total droplets	Low fraction of droplets contain successful PCR amplification	Ensure the correct amount of gDNA is added to each sample. Check that the correct concentration of primers and probe was used, optimise if necessary.
		Droplet generation failure	Ensure correct dilution of probe master mix and correct total volume. Ensure all wells in a column contain probe mastermix or buffer (not water, or empty).
96)	Gel electrophoresis of the IVT mRNA shows no band, smear, or incorrectly sized band	Transcription failure	Ensure input DNA has a 5′ T7 promoter and 1 μg of high quality DNA was used as the template. Use fresh enzymes.
Box 3	Gel electrophoresis of the IVT mRNA shows no band, smear, or incorrectly sized band	RNAse contamination	Repeat IVT protocol with RNase free technique