Volume of lentiviral particles required for screening with CRISPRmod CRISPRa lentiviral sgRNA whole genome pooled screening libraries

Volume of lentiviral particles at 5 × 10⁸ TU/mL in HEK293T cells required for the indicated fold representation, biological replicates, and sgRNAs/gene, assuming experimental cells have same relative transduction efficiency as HEK293T. The volume of lentiviral particles required for your screen can be calculated using the CRISPR pooled screening protocol tracking worksheet

Gene activation screening workflow using the CRISPRmod CRISPRa lentiviral sgRNA pooled screening library platform

A dCas9-VPR-expressing stable cell line (mixed or clonal cell population) is first generated with CRISPRmod CRISPRa Lentiviral dCas9-VPR particles by selection with blasticidin. These cells are then transduced with a CRISPRmod CRISPRa lentiviral sgRNA pooled library and selected with puromycin. Transduced cells are split into reference and experimental populations for application of a selective pressure and/or phenotypic selection. Genomic DNA is then isolated from the reference and experimental populations of transduced cells and sgRNA constructs within the isolated gDNA are amplified, indexed, and prepared for high-throughput sequencing on an Illumina platform using the Dharmacon™ NGS Library Prep Kit. The integrated sgRNA sequences in both reference and experimental samples are identified and relative abundance compared. sgRNA constructs that are enriched or depleted are identified as hits to be confirmed and studied further using individual CRISPRmod CRISPRa Lentiviral sgRNAs in additional phenotypic and/or biochemical assays.

Transductions with CRISPRa lentiviral sgRNA in different dCas9-VPR cell lines show robust transcriptional activation

Figure 1: U2OS, HEK293T, MCF 10A and K562 stably expressing integrated CRISPRa dCas9-VPR were plated at 10,000 cells/well and transduced with CRISPRa sgRNA lentiviral particles targeting POU5F1 or TTN at a MOI of 0.3 to obtain cells with a single integrant. Cells were selected with 2 µg/mL puromycin for 4 days prior to analysis with RT-qPCR. The relative expression of each gene was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control

CRISPRmod CRISPRa lentiviral sgRNA libraries undergo rigorous production and QC for high quality pooled screening 

Figure 2: High quality pooled screening begins with rigorous lentiviral pooled library production. CRISPRmod CRISPRa lentiviral sgRNA whole genome pooled libraries comprising 4 sgRNAs per gene (left) and 8 sgRNAs per gene (right) were produced and the quality of the plasmid DNA library was verified by next-generation sequencing (NGS). Counts per million mapped reads were obtained to determine the percent-recovery of input sgRNAs 98.9% (left) and 99.8% (right) and that the distribution of 90% and 70% of the sgRNAs in the pool are within 8.8 (left), 10.6 (right) and 3.4 (left), 4.0 (right) fold of each other, respectively, indicating high sgRNA recovery and uniform distribution of sgRNAs if both the 4 sgRNA per gene and 8 sgRNA per gene whole genome libraries. For more information on skew ratios for genome-wide libraries, please see: https://pmc.ncbi.nlm.nih.gov/articles/PMC10797759/.

Highly concordant hits observed between whole-genome CRISPRa screens performed using the CRISPRmod All-in-one dCas9-VPR platform and Synergistic Activation Mediator (SAM) systems

Figure 3: Highly concordant resistance hits using a CRISPRmod CRISPRa Human All-in-one lentiviral sgRNA Whole Genome Pooled Library (A) or the three vector Synergistic Activation Mediator System (B) in A375 melanoma cells treated with vemurafenib (PLX-4032) for 16 days. DrugZ screen analysis showing log2 fold enrichment of each gene and associated p-values; significantly enriched genes (positive Log2 Fold Change) drive resistance to the drug treatment indicating these genes potentially act as drug antagonists. Highlighted hits were identified with both technologies and have been previously validated by CRISPRa screening (Konermann et al., 2015).

Application example: High-throughput pooled CRISPR screening with high-capacity single cell analysis can be effectively achieved with gene family pathway libraries. These libraries are now available in CRISPRa formats as well, supporting the data and conclusions observed below for CRISPRko screening with a protein kinase sgRNA library of the same gene composition.

Figure 4: A pooled lentiviral library comprised of 3240 Edit-R sgRNAs targeting 760 human kinases, a small panel of essential genes, and 100 non-targeting controls (NTCs) was transduced into Strict-R inducible Cas9-expressing U2OS and A549 cell lines at MOI of 0.3 and 300- fold sgRNA representation. At 24 hours post-transduction, cells were selected with 2 μg/mL puromycin for 4 days. The reference (T0) sample was harvested, and matched samples were stimulated with 500 ng/µL doxycycline/ 500 nM Shield1 or cultured in standard growth media for 8 days. Genomic DNA was harvested from the T0, uninduced and induced samples and PCR-amplified for high-throughput sequencing on an Illumina platform. Log2 fold change of induced U2OS cells compared to T0 is plotted vs. –log10 MAGeCK P-value. The sgRNAs that were significantly (FDR ≤ 0.10) higher and lower abundance that also showed > |2|-fold abundance change are in green and purple, respectively. NTC abundance is starred in red, and the green line indicates zero-fold abundance change. See our full scientific poster for more details.