Table 1 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
47	Poor enrichment of CD4+ T cells	Inefficient isolation of PBMCs from whole blood or impure PBMCs	Perform isolations within 24 h of blood draw Increase dilution of blood samples in Step 21 Handle centrifuged samples with care and turn off the centrifuge brake to ensure that a clean PBMC buffy coat forms in Step 23 Wash the PBMCs with PBS-EDTA one extra time after Step 28
		Inefficient binding and removal of CD4− cells from PBMCs	Recount the PBMCs to ensure a correct concentration of input cells in Step 31 Ensure complete mixing of antibody and beads during Steps 35–37
		Donor has low T-cell counts Inefficient CD4 staining	Start over with fresh blood from a new donor Increase antibody concentration during incubation or stain fewer cells in Step 47 Wash twice with MACS buffer before staining Incorporate a compatible live-dead cell stain to remove confounding signals
	Inefficient T-cell activation	Poor CD3 adherence to the surface of the plate	Use non-treated plates and increase either CD3 concentration or incubation time in Step 18 Switch to either bead-based or small molecule-based activation protocols
		Donor T cells are exhausted and cannot be activated	Start over with fresh blood from a new donor
		Inefficient CD25 staining	Increase antibody concentration during incubation or stain fewer cells in Step 47 Wash cells twice with MACS buffer before staining Incorporate a compatible live-dead cell stain to remove confounding signals
66, 73	CRISPR-Cas9 RNPs show little activity in positive controls	Donor cells are not sufficiently activated	Cells should be nucleofected 48–72 h after activation and should be >90% CD25+ in Step 47. If not, reactivate or start over with fresh blood from a new donor
		Guide RNA stocks were suspended >6 months earlier or frozen-thawed multiple times	Reorder guide RNA
		crRNPs were generated >6 months earlier or frozen-thawed multiple times	Resynthesize crRNPs
		Cas9 precipitated during synthesis	Preheat Cas9, tracrRNA, and guide RNA to 37 °C before mixing in Step 17 If a precipitate is visible, mix gently by pipette or flicking until precipitant re-enters solution in Step 17
		Too many cells or too few crRNPs were added to the nucleofection mixture	Repeat nucleofections with fewer cells or more crRNPs per reaction
		Arc error during nucleofection of the positive=control well	Repeat nucleofections, making sure to avoid air bubbles, to pipette to the bottom of the cuvette, and to add no more than 20 μL of suspension per well in Step 56
		Cells were harvested for validation before protein turnover	Wait 72–96 h before harvesting protein or genomic DNA in Step 67
	Positive controls are validated, but experimental CRISPR-Cas9 RNPs do not work	Guide RNA does not target a translated exon	Double-check the intron-exon structure of the targeted gene, with special attention to alternative isoforms in Steps 1–9
		Guide RNA does not cut the target site	Not all guides will be successful. Redesign and order an additional guide RNA targeting the desired locus
		Targeted locus is not accessible to editing	Some loci appear resistant to editing in some cell types. Try multiple guide RNAs or alternative cell types or lines
73	TIDE analysis does not work	Unclear chromatograms prevent deconvolution	Adjust PCR thermocycler conditions to amplify a single amplicon in Step 70. Verify the success of adjustments by gel electrophoresis Design and order alternative primers in Step 68 if necessary Ensure clean DNA preparations after the purification performed in Step 71 by spectrophotometry
74–108	Low infection rate (<1%)	Poor yield during virus preparation	Check transfection efficiency in Steps 78–83 with a GFP or reporter plasmid under identical conditions and adjust if necessary Ensure purity and correct sequence of the HIV molecular clone by full-vector sequencing and spectrophotometry During concentration, make sure the mixture is cloudy after 4 °C incubation in Step 88, before pelleting of virions. If it is not, add more PEG-6000 and incubate for 2 h to promote precipitation Verify virus titer by p24 ELISA or infection of an easy-to-infect cell line as described in Step 93
		The molecular clone used for virological assays is not appropriate for replication in primary cells	Choose an alternative molecular clone
		The cells are not fully activated	Stimulate again and repeat infection
		The donor is not susceptible to HIV infection	Start over with a new blood sample from a different donor
		Donor cells are only partially susceptible to infection	Infect with more virus in Step 100 or increase infection rates with alternative infection techniques such as spinoculation Allow more time for infection to spread between cells in Steps 100–106