Volume of lentiviral particles required for screening with CRISPRmod CRISPRi 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 repression screening workflow using the CRISPRmod CRISPRi lentiviral sgRNA pooled screening library platform.

A dCas9-SALL1-SDS3-expressing stable cell line (mixed or clonal cell population) is first generated with CRISPRmod CRISPRi Lentiviral dCas9-SALL1-SDS3 particles by selection with blasticidin. These cells are then transduced with a CRISPRmod CRISPRi 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 CRISPRi Lentiviral sgRNAs in additional phenotypic and/or biochemical assays.

CRISPRi lentiviral sgRNA is well suited for long term repression

Figure 1: U2OS and A549 cells stably expressing integrated dCas9-KRAB or dCas9-SALL1-SDS3 were plated at 50,000 cells/well in 24 well plates and transduced with CRISPRi sgRNA expressing lentiviral particles targeting PPIB or SEL1L at a MOI of 0.3 to obtain cells with a single integrant. Cells were selected with 2 µg/mL puromycin for 5 days, split into two populations, and allowed to recover for an additional day. At 7 days post-transduction one population was harvested for RT-qPCR analysis. The replicate populations of cells were cultured for 7 additional days before harvest. Total RNA was isolated from harvested plates and relative gene expression was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.

CRISPRmod CRISPRi 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 CRISPRi 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 99.8% (left) and 99.5% (right) and that the distribution of 90% and 70% of the sgRNAs in the pool are within 9.1 (left), 8.3 (right) and 3.8 (left), 3.7 (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/.

Assessment of essential and non-essential gene dropout with the All-in-one dCas9-SALL1-SDS3 CRISPRi system, CRISPRi KRAB system and CRISPRko system

Figure 3: To assess the efficacy of the whole genome loss-of-function screens, we analyzed a subset of guide RNAs that target a group of 684 core essential genes (CEG) and 1000 non-essential genes (NEG) for gene knockout (Cas9) or repression (either dCas9-KRAB or dCas9-SALL-SDS3) in the reference population (DMSO-treated only) of A375 melanoma cells. By assessing the degree of essential “gene dropout” for sgRNA targets that become depleted relative to an initial timepoint, one can establish an unbiased measure of platform performance. Violin plots illustrate the degree of CEG and NEG dropout in the three loss-of-function platforms. Each screening platform exhibits significant essential gene dropout, and neutral non-essential gene behavior, serving to validate the incorporated whole-genome screen in each case. See our full Application Note for more details.

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 CRISPRi 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.