Genome-wide CRISPR knockout screen in Huh7.5.1 human hepatocytes for the identification of host factors of hepatitis A virus
ABSTRACT: A knockout cell library in Huh7.5.1 cells was generated by introducing a genome-scale CRISPR library (GeCKOv2, Addgene #1000000049) and subjected to hepatitis A virus infection (HM175/18f) to isolate virus-resistant mutant cells. Genomic DNA was isolated from the original and virus-selected mutant cell populations and abundance of guideRNA encoding sequences were measured by sequencing on an Illumina NextSeq (High Output).
Project description:We performed genome-wide CRISPR KO screens in human Huh7.5.1 cells to select for mutations that render host cells resistant to viral infection by SARS-CoV-2, human coronavirus 229E and OC43.
Project description:H1-HeLa cells were stably transduced with lentiCas9-Blast (Addgene, Plasmid #52962) and subsequently selected using blasticidin to generate constitutively expressing Cas9 H1-HeLa cells. A single Cas9-expressing H1-HeLa clone was then transduced with lentivirus without a selection marker to stably express CDHR3 C529Y (H1-HeLa+CDHR3). A single CDHR3-expressing H1-HeLa clone was then chosen based on RT-qPCR of CHDR3 expression and RV-C15 RNA levels for mutagenesis. 300 million of the H1-HeLa cells constitutively expressing CDHR3 and Cas9 were transduced with the lentiGuide-Puro from the GeCKO v2 library at a MOI of 0.3. Cells were selected using puromycin and heterogeneous H1-HeLa knockout cell populations were subsequently pooled together. The CRISPR genetic screens were started 10 days post transduction. >1000-fold coverage of mutagenized cells (libraries A and B) was infected with either RV-C15 (MOI=1 PFU/cell) or EV-D68 Missouri (MOI=1 PFU/cell). RV-C15 infection was repeated for an additional round at 6 days post-infection. As soon as appearance of visibly viable colonies was observed, populations of virus-resistant cells were pooled and harvested. Uninfected starting populations of mutagenized cells were used as the unselected reference. Total genomic DNA from both virus-resistant and uninfected cells was respectively extracted using QIAamp DNA Mini Kit (Qiagen). The inserted guide RNA sequences were retrieved from the genomic DNA by PCR amplification. The PCR products were then purified and subjected to NextSeq platform (Illumina) next-generation sequencing.
Project description:West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen using CRISPR/Cas9. HEK 293FT cells were infected with lentivirus expressing sgRNAs and then transfected with a Cas9 expressing construct. WNV infection killed most cells during a 12d selection. Survivor cells were harvested, from which DNA was isolated. The sgRNAs integrated in genome of survivor cells were amplified with PCR. The PCR product was sequenced with Illumina MiSeq to profile the sgRNA population in the survivor cells. Three replicates were conducted. Similarly, a second round of screen was conducted. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J2, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Examination of sgRNA populations in survival 293FT cells
Project description:Splenocytes, peripheral blood cells, peritoneal cells and tumor cells were washed with PBS and were counted. Cells were centrifuged at 400 g for 5 min and supernatant was removed. Cells pellet (< 3 x 106 cells) was resuspend in 350 µl of RLT buffer (Qiagen). RNA was extracted with RNeasy Mini kit (Qiagen) according to manufacturer’s protocol. mRNA library preparation was realized following manufacturer’s recommendations (KAPA mRNA HyperPrep ROCHE). Library purity/integrity were assessed using an Agilent 2200 Tapestation (Agilent Technologies, Waldbrunn, Germany). Final 7 samples pooled library prep were sequenced on Nextseq 500 ILLUMINA with MidOutPut cartridge (2x130Millions of 75 bases reads), corresponding to 2x18Millions of reads per sample after demultiplexing.
Project description:The goal of this study is to investigate the differential transcripted genes affected by CRISPR induced endoglin knockout in PASMC cells. Total RNA was purified from NTC or ENG-/- PASMC cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quality and concentration were assessed with Agilent Tapestation 200 (Agilent Technologies) and Qubit 2.0 (ThermoFisher Scientific). ~250-500 ng RNA were used for library construction. The NGS libraries were constructed using the KAPA Stranded mRNA-Seq Kits (KapaBioSystems). mRNA was captured using magnetic oligo-dT beads and 1st strand cDNA was synthesized using random priming. In order to preserve strand-specificity, 2nd strand synthesis, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), was marked by dUTP incorporation. cDNA framents were A-tailed by adding dAMP to the 3'-ends of the dscDNA library fragments. dsDNA Illumina TruSeq "forked” adapters 3'-dTMP overhangs were then ligated to A-tailed library insert fragments. Each of the six libraries were ligated with a unique Truseq 6bp barcode. Library fragments were amplified using the KAPA HiFi HotStart polymerase. The strand marked with dUTP was not amplified, allowing strand-specific sequencing. Fragment length and library quality was assessed on a 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies). Libraries were diluted to 10nM and pooled at equimolar ratios. The pool was then diluted to 2nM and denatured in NaOH following Illumina recommendations. 10pM of denatured library pool was loaded in one HiSeq lane and flowcell was clustered on the Illumina C-bot. 5% PhiX control was spiked-in. The flowcell was sequenced on a HiSeq 2500 V4 chemistry with 50bp Single read protocol. Data was demultiplexed and Fastq files were generated using BcptoFastq 1.8.4 script provided by Illumina.
Project description:Double-strand DNA breaks (DSBs) continuously arise and are a source of mutations and chromosomal rearrangements. Here, we present DSBCapture, a sequencing-based method that captures DSBs in situ and directly maps these at single nucleotide resolution enabling the study of DSB origin. DSBCapture shows substantially increased sensitivity and data yield compared to other methods. Employing DSBCapture, we uncovered a striking relationship between DSBs and elevated transcription within nucleosome-depleted chromatin. 6 library samples, 75 base pairs (50 bp for the EcoRV library) custom protocol (DSBCapture or BLESS) sequenced as paired-end reads on Illumina NextSeq 500 (MiSeq for EcoRV library): 1 replicate for the EcoRV library, 1 replicate for the library coming from the U2OS AID-DlvA cell line with AsiSI restriction enzyme, 2 replicates for the BREAk-seq NHEK libraries and 2 replicates for the BLESS NHEK libraries. 4 RNA-Seq library samples from HEK Gibco cells, single-end sequencing on the Illumina NextSeq 500, 75 base pairs.
Project description:Little is known about genes that promote melanoma cell growth and proliferation. siRNAs may be used to address the role of individual genesin these processes. RNAi library screens were used in the past to gain a comprehensive overview of all genes involved in cell growth, proliferation, migration and other cellular processes. A large-scale loss-of-function screen for eight different melanoma cell lines was performed using a pooled lentiviral shRNA library (GeneNet Human 50K lentiviral shRNA Library,cat#SI206B-1, System Biosciences) to identify genes relevant for melanoma cell growth and proliferation. shRNAs that lead to cell death or reduced growth of transduced melanoma cells are negatively selected and thereby underrepresented in the final cellular shRNA pool and vice versa. The shRNAs of the shRNA library (3-5 per gene) have complementary sequences to probes on the custom Affymetrix microarray HG-U133Plus2 and were analysed using this array. Well-known melanoma cell lines SK-Mel-103, A375, SK-Mel-147, SK-Mel-19, SK-Mel-28, SK-Mel-29, SK-Mel-5, WM3523cln6 were transduced with the lentiviral shRNA library and grown for 10 days under puromycin selection (day 10), control cells of respective cell lines were transduced and frozen immediately after transduction and genomic integration of shRNAs (day 0). Totel DNA was extracted and genomically integrated shRNAs were hybridized to Affymetrix microarrays (HG-U133Plus2.0 array).
Project description:Host cells produce interferon (IFN) in response to viral infections. Secreted interferon results in the transcription and production of hundreds of interferon-stimulated genes (ISGs). A genome-wide CRISPR screen using IFN alpha-treated Huh7.5 cells was performed to determine which ISGs were required in order for host cells to suppress yellow fever virus (YFV) infection. Overall design: Human Huh7.5 cells were transduced with a human CRISPR library in a single replicate at 900X library coverage, treated with IFN alpha, infected with YFV-17D-venus, and GFP-positive cells that escaped IFN-alpha suppression of YFV-17D-venus infection were collected by FACS.
Project description:Identification of factors to inhibit influenza virus infection Overall design: CRISPR activation technology was used to identify genes that rendered the cell refractory to infection. The complete sgRNA library (plasmid sample) were packaged into lentivirus and used to transduce A549 cells (transduced sample). In three independent biological replicates, the transduced cells were infected with influenza virus, and the cells that were refractory to infection were collected and the sgRNAs enriched in those populations were sequenced (samples A, B, and C).