Why are there four sgRNAs per gene and not eight?
Why are there four sgRNAs per gene and not eight?
Smaller libraries such as the Guide-it CRISPR Genome-Wide sgRNA Library System have a higher percentage of active and specific sgRNAs, which streamlines the screening process and makes screening far more cost-effective. The four guides per gene in our library are designed to be highly active since they are chosen on the basis of an algorithm that ensures high on-target activity (Doench et al. 2016) and improved sgRNA scaffold design (Chen et al. 2013).
Libraries that use less well-designed guide RNAs may provide eight guides per gene to counteract the lower percentage of active and specific sgRNAs. Users of libraries with eight guides need to screen double the number of cells, which represents a significant amount of extra work (more virus production, more cells to transduce) and additional sequencing, analysis, and cost.
ReferencesDoench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR Cas9. Nat. Biotechnol. 34, 184–191 (2016).
Chen, B. et al. Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System. Cell 155, 1479–1491 (2013).