Question: Which Capture Sequence and sgRNA integration site should I use for 3' CRISPR Screening experiments?
Answer: When designing sgRNAs for use with the 3’ Single Cell Gene Expression and CRISPR Screening solution, each sgRNA should be engineered to contain either Capture Sequence 1 (CS1) or Capture Sequence 2 (CS2); integrated either at the 3’ end of the sgRNA or within the sgRNA hairpin structure. These sequences and integration sites are described in our Technical Note: Guide RNA Specifications Compatible with Feature Barcoding for CRISPR Screening.
To determine which Capture Sequence and integration site is optimal for your cell type, we recommend performing a pilot experiment using the MilliporeSigma Optimization Kit. This kit allows you to test each of the four possible configurations:
- CS1 integrated at the 3’ position of the sgRNA
- CS1 integrated within the sgRNA hairpin
- CS2 integrated at the 3’ position of the sgRNA
- CS2 integrated within the sgRNA hairpin
During our in-house tests on four human cell lines (A375, A549, U2OS, SKOV3), we found that CS2 in the hairpin position consistently produced the best capture efficiency. In other tests with K562 and Jurkat cells, we observed robust capture efficiency with CS1 in the hairpin position, although not all possible configurations were tested. We recommend performing pilot experiments to determine which configuration leads to optimal capture efficiency in your cell type and experimental system.
The effect of the Capture Sequence and integration site on guide efficacy should also be considered. It is important to note that the Capture Sequence and location that give the best capture efficiency do not always result in the best guide efficacy. For the cell lines mentioned above (A375, A549, U2OS, SKOV3), CS2 in the hairpin position resulted in guides with strong efficacy, though not always the absolute strongest of all possible configurations. In K562 cells, Replogle et al (2020) demonstrated that the activity of a guide targeting GFP was compromised when CS1 was inserted at the 3’ end but was not compromised with alternative configurations. We recommend performing pilot experiments, such as assessing knockdown efficiency by qPCR, to assess guide efficacy in your experimental system.
For 5' CRISPR Screening, direct capture of the gRNA sequence is enabled without modification of the CRISPR-Cas9 library with CS1 or CS2.
Products: Single Cell Gene Expression