Table 2.

Common problems, possible causes, and solutions to address the problems.

Problem	Possible Cause	Solution
No lymphoblastoid cells alive after selection	Transfection is unsuccessful	Check that 293FT cells produce green fluorescence signal 24h after transfection of pXPR_011.
		Check the concentrations of the packaging (at least 1 μg/μL) and lentiviral expression constructs (at least 100 ng/μL). Perform maxiprep if necessary.
	Packaging of lentiviruses is unsuccessful	Use low-passage (<20 passages) 293FT cells for transfection.
No colonies present after sgRNA cloning	Oligo annealing is unsuccessful	Ensure that the sgRNA oligos have been diluted to the correct concentration. Make sure that the correct pairs of oligos have been used in the reaction, especially when multiple sgRNAs are being cloned at the same time.
	Ligation is unsuccessful	Keep T4 DNA ligase at -20°C and minimize prolonged exposure to higher temperatures. Perform ligation reaction longer than 16 hours.
Too many colonies (> 100) present after sgRNA cloning	Linearized vector is contaminated with uncut vector	Perform vector digestion again, incubating for longer periods or using more restriction enzyme. Ensure that cut and uncut forms of the vector are sufficiently separated on the agarose gel.
Quantitative RT-PCR shows no change to gene expression levels in sgRNA-transduced CRISPRi-competent cells	Alternative promoter usage with the use of a qPCR primer pair that amplifies a region common to the transcript isoforms	Use a primer pair that amplifies as close as possible to the target promoter’s transcription start site.
Quantitative RT-PCR shows no change in gene expression levels in sgRNA-transduced cells	sgRNA sequence is incorrect	Sequence the sgRNA expression construct to ensure that the targeting sequence is correct.
Immunoblotting and/or flow cytometry shows no change to protein expression levels in sgRNA-transduced cells	Antibody does not detect the target protein	Perform conventional CRISPR, followed by immunoblotting to see if the band corresponding to the detected protein disappears. If the band persists, purchase a new antibody and test it against the same whole-cell lysate.