CRISPR Libraries

Dual-gRNA libraries are far more powerful than single-gRNA libraries for knockout screens because the introduction of large deletions by these libraries can have much higher efficiencies in generating loss-of-function mutations. Each CRISPR vector in a dual-gRNA library contains a pair of gRNAs targeting the same gene. When introduced into Cas9-expressing cells, each vector can produce two cuts on the same target gene. Attempts by cells to repair the broken ends of the two cut sites would typically lead to a large deletion spanning the two sites. The two cut sites are designed to flank a functionally important region of the target gene such that a deletion spanning them would most likely lead to the loss of gene function.

For typical knockout screens, a pooled CRISPR library can be constructed in the format of either 1-vector or 2-vector systems. In 1-vector systems, Cas9 or Cas9 variant is co-expressed with gRNA(s) from the same vector, while in 2-vector systems, Cas9 or Cas9 variant and the gRNA(s) are expressed from two separate vectors. Alternatively, in 2-vector systems, gRNA-expressing vectors can be introduced into cell lines that stably express Cas9. The advantages of using the 1-vector systems are that it avoids co-transfection/transduction of two different vectors into cells and it is not limited to the availability of a Cas9-expressing stable cell line. However, it is less versatile and can be less efficient in cloning and virus packaging than the 2-vector systems.

VectorBuilder follows the algorithm utilized in CRISPR library design (CLD) to calculate specificity scores for gRNAs. Briefly, for a given gRNA intended to target an N(20)NGG sequence in a species, we search for all potential off-target sites in the genome of that species that have ≤3 mismatches with the target sequence. For each potential off-target site identified this way, a single off-target score is calculated. Scores for all the off-target sites are then used in aggregate to calculate the final specificity score of the gRNA, which is between 0 and 100, with higher values indicating greater targeting specificity.

Please note that specificity scores are only a rough guide. Actual targeting efficiency and specificity could depart from what the scores predict. gRNAs with low scores may still work well.