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. Author manuscript; available in PMC: 2020 Dec 1.
Published in final edited form as: Curr Protoc Mol Biol. 2019 Dec;129(1):e111. doi: 10.1002/cpmb.111

Table 3:

Common problems encountered, potential causes, and proposed solutions

Problem Potential cause Solution
Low integration efficiency.

  1. Low transfection efficiency.

  2. Damaged or degraded plasmid DNA.

  1. Ensure that cells are doubling. approximately once per day prior to transfection.

  2. Perform fresh minipreps of pBS130 and plasmid library prior to transfection.
Cell doubling rate does not consistently decrease following cytotoxin treatment. Cytotoxin may be losing activity. Use Support Protocol 1 to confirm cytotoxin effect on naïve cells periodically during cytotoxin exposure to the CRISPR library to ensure that it has not lost activity; replace as needed.
Cell-lines crash during expansion

  1. Cytotoxin treatment had too great an effect on doubling.

  2. Contamination occurred.

  1. Lower cytotoxin level and repeat.

  2. Discard and replace all cell culture reagents and repeat. For general advice about Drosophila cell culture, see Luhur et al. (2019).
A few sgRNAs targeting different genes dominate reads

  1. Insufficient cell coverage of CRISPR library leading to artifactual “bottleneck” in cell population

  2. Insufficient template DNA during PCRs led to “bottleneck” in PCR products.

  1. Increase coverage of cells per passage.

  2. Increase total volume of PCRs used in PCR1 reaction.