transEDIT-Dual sgRNA CRISPR KO Whole Human Genome Arrayed Library –

Dual sgRNA Arrayed CRISPR KO Library: Targeting with Multiple sgRNAs Results in Predictable Genomic Scars
(A) Schematic map of the lentiviral, dual-sgRNA expression vector with relevant features highlighted. hU6, human U6 promoter; cU6, chicken U6 promoter; hsgRNA, human U6-promoterdriven sgRNA; csgRNA, human U6-promoterdriven sgRNA; HTS, high-throughput sequencing adapters; SFFV, spleen focus-forming virus promoter. (B) sgRNA pairing algorithm used to design five targeting constructs for a gene. The top 20 sgRNAs for each gene are filtered to a set of 10 to reduce the probability of off-targeting effects. Pairs within these 10 are then scored using the set of heuristics defined in Figure S2. The resultant pairing matrix is then used as input for a maximum-weighted matching algorithm to define a final set of 5 sgRNA pairs. (C) Paired-end sequencing analysis of genomic scars left after dual-CRoatan NEG-targeting constructs have been infected into A-375 and K-562 cells. hsgRNA and csgRNA indels are where only one of the two targeted regions shows mutational burden in an HTS fragment. hsgRNA and csgRNA indel counts represent cases where both targets have indels, and fragment deletions are where the region between the two targets is deleted. (D) Analysis of the genomic scars described in (C) that correspond to fragment deletions between two sgRNA target sites. The top ten most frequent deletions are shown with their corresponding rate of occurrence, as measured by their average frequency in infected A-375 and K-562 cells. Scars that result from exact deletion of the doublestrand-break-flanked fragment are annotated as DSB-DSB deletions. Scars where, in addition to the fragment deletion, other bases are inserted or deleted are annotated as non-DSB-DSB deletions.