Does every sgRNA in the library work?

It is impossible to test all 76,456 sgRNAs functionally. However, the target sequences cloned into the Guide-it Genome-Wide sgRNA Library are the same as those described by Doench et al. in 2016. In this paper, the authors created and modified their Brunello algorithm based on real screening and activity data from positive and negative screens. At Takara Bio we verified the editing activity of a [...]

Why is guide RNA scaffold design an important feature of the library?

Two parts of every guide RNA are essential for its activity: the 20-nucleotide target sequence which determines the specificity to the target gene and the looped-structured scaffold that determines how well the guide RNA binds to the Cas9 protein. The sgRNA scaffold encoded by the Guide-it library vector pLVXS-sgRNA-mCherry-hyg uses an optimized sequence for improved editing efficiency. This [...]

What is the Brunello algorithm that was used to create the library?

The target sequences cloned into the Guide-it Genome-Wide sgRNA Library are the same as described by Doench et al. in 2016. In this paper, the authors created and modified their algorithm based on real screening and activity data from positive and negative screens. They named their human library ""Brunello."" ReferenceDoench, J. G. et al. Optimized sgRNA design to maximize activity and minimize [...]

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. [...]

Why are there 76,612 different guides in the Guide-it CRISPR Genome-Wide sgRNA Library?

19,114 genes targeted by four sgRNAs each = 76,456 sgRNAs (plus 156 control sgRNAs) = 76,612 sgRNAs Gene editing [...]