4. Protocol Overview

Cellecta’s CRISPRtest™ Essential-Gene Cas9 Activity Kit allows you to assess the level of Cas9 activity in your human cell line using the CRISPRtest virus which contains two pre-mixed lentiviral-packaged vectors:

• One vector containing a green (GFP) or blue fluorescent protein (BFP) marker (depending on which version of the kit you are using) and an sgRNA sequence targeting an essential gene in human or mouse cells (also depending on the version of the assay you are using).
• A second vector containing a red fluorescent protein (RFP) marker and a non-targeting sgRNA.
  

Transduction of the CRISPRtest virus into cells will result in an initial ratio of GFP (or BFP) cells to RFP cells. After cell growth in the presence of active Cas9, the ratio of GFP (or BFP) cells to RFP cells will decrease proportionally to Cas9 activity.

CRISPRtest Essential-Gene Cas9 Activity Kit Workflow

Cas9 cell line and Parental cells are transduced with the CRISPRtest Virus. At Day 3 and Day 10 after transduction, a portion of the cells are analyzed by flow cytometry to determine GFP:RFP ratios. These ratios are then used to calculate the percentage knockout which is an assessment of Cas9 activity