Perturb-Seq or CROP-Seq screens make use of single-cell RNA-Sequencing in conjunction with a pooled CRISPR library to identify transcriptional changes and, by implication, activation or deactivation of cellular pathways related to phenotypic changes produced by specific sgRNA-mediated gene knockouts.
These sort of sgRNA-induced expression analysis screens with pooled sgRNA require the coupling of cell-specific transcriptome profiling with the identification of the specific sgRNA sequences expressed by each cell in a single experiment. There are a couple of approaches to extract both of these data sets using any of the several single-cell expression profiling platforms current available from companies such as 10X Genomics, Bio-Rad, Dolomite Bio, and others.
One approach is to design the pooled CRISPR library so that the sgRNA expression cassette is inserted into the 3’-region of an expressed selection marker gene on the vector. With this configuration, the guide is expressed near the poly-A+ tail of a standard mRNA transcript so it is reverse-transcribed and detectable in the cDNA generated as part of the RNA-seq protocol. This variation is the CROP-Seq approach. Cellecta has developed the pScribe vector to make use of this approach with its sgRNA libraries.
However, another approach is to capture the sgRNA directly, even when it is not expressed as part of an mRNA transcript. For this approach, another method is used to directly capture the small expressed sgRNA molecules present in extracted RNA.
10X Genomics, one of the single-cell platform companies, has designed cell capture beads for its platform that include unique barcode sequences, as well as the standard poly-dT sequences. Beads with these additional barcodes enable the retrieval from each cell of molecules modified with complementary sequences (i.e., “barcoded features”), while at the same time, the poly-dT sequences bind poly-A tails and capture mRNA for reverse transcription as usual. This approach enables correlation and identification of the captured, barcoded molecules with the transcript profile from each cell.
10X Genomics describes how its Feature Barcoding approach can be used to identify sgRNA present in cells if the guide is modified so it contains the complementary capture sequence for the Feature Barcodes. This approach, then, requires a specially designed sgRNA structure that has an additional insert to enable it to bind to the one of the Feature Barcodes on the bead.
Below, you can see two options they recommend for modifying the sgRNA to insert this capture sequence into the sgRNA. While previously published data indicated the additional sequence could be inserted into the standard sgRNA structure without affecting its efficacy, we thought it prudent to also confirm for ourselves that this additional sequence does not alter sgRNA activity. In particular, because we typically utilize an sgRNA design that incorporates the HEAT tracr design which we have found more efficacious for knockout and library screening.
You can see the data below comparing sgRNA containing 10X Genomics' recommended capture sequences with our standard sgRNA. The capture sequence insertions do not affect performance of our HEAT-modified sgRNA.
Left: Various options of capture sequences recommended by 10X Genomics for insertion into the sgRNA to be used with their Feature Barcodes
Right: Cellecta made and tested all 4 capture sequence variations and confirmed that the insertions do not affect performance of our HEAT-modified sgRNA