Sequencing and analysis of CRISPR screens

Genomic DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen 69504) per the manufacturer’s instructions. gRNAs were identified in our populations using PCR primers with Illumina adapters. Genomic DNA from a number of cells corresponding to the equivalent of 250-fold library coverage was used as template for PCR (20 million cells for knockout screen, 15 million cells for activation screen). 10μg of gDNA was used in each PCR reaction along with 20μl 5X HiFi Reaction Buffer, 4μl of P5 primer, 4μl of P7 primer, 3μl of Radiant HiFi Ultra Polymerase (Stellar Scientific), and water. The P5 and P7 primers were determined using the user guide provided with the CRISPR libraries (https://media.addgene.org/cms/filer_public/61/16/611619f4-0926-4a07-b5c7-e286a8ecf7f5/broadgpp-sequencing-protocol.pdf). The PCR cycled as follows: 98°C for 2min before cycling, then 98°C for 10sec, 60°C for 15sec, and 72°C for 45sec, for 30 cycles, and finally 72°C for 5min. After PCR purification, the final product was Sanger sequenced to confirm that the guide region is present, followed by qPCR to determine the exact amount of PCR product present. The purified PCR product was then sequenced with Illumina HiSeq 2500 single read for 50 cycles, targeting 10 million reads.

Next, the sequencing results were analyzed bioinformatically. First, the gRNA representation was analyzed using the custom python script provided (count_spacers.py). [56]. The difference between the number of guides present in each ATRi condition compared to control condition was then determined. Specifically, one read count was added to each gRNA, and then the treatment reads were normalized to no treatment. Finally, the values found were used as input in the Redundant siRNA Activity (RSA) algorithm [27, 57]. For RSA, the Bonferroni option was used and guides that were 2-fold enriched in treatment compared to no treatment were considered hits. This analysis method allows for the identification of genes that are upregulated in one population (VE822 or AZD6738) compared to control. Hits are determined by the amount of gRNA sequences present in the population and the number of guides per gene present. Furthermore, p-values are determined by the RSA algorithm for the genes that are most enriched in the test populations compared to the control. VE822 and AZD6738 top hits can then be compared. Biological pathway analysis of the top hits was performed using Gene Ontology [58, 59].