Why is guide RNA scaffold design an important feature of the library?
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 modified scaffold maximizes the binding affinity between the sgRNA and the Cas9 endonuclease (Chen et al. 2013), which increases editing efficiency, and compared to some traditional scaffolds used in other libraries, provides a greater chance that each guide is active.
ReferenceChen, B. et al. Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System. Cell 155, 1479–1491 (2013).