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Sgrna library

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The SgRNA library is a collection of short guide RNA (sgRNA) sequences designed for use in CRISPR-Cas9 gene editing experiments. The library provides a comprehensive set of sgRNA targeting various genes, allowing researchers to efficiently and systematically investigate gene function.

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Lab products found in correlation

Hiseq 2500, by Illumina (2 mentions) Puromycin, by Thermo Fisher Scientific (2 mentions) Accutase, by Innovative Cell Technologies (2 mentions) Blasticidin, by Solarbio (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) Megashortscript kit, by Thermo Fisher Scientific (1 mentions) Nextseq, by Illumina (1 mentions) Puromycin, by Addgene (1 mentions) Tianamp genomic dna kit, by Tiangen Biotech (1 mentions) Dnase 1, by Thermo Fisher Scientific (1 mentions) Pspax2, by Addgene (1 mentions)

Close spelling variants of this product

GeCKO v2 sgRNA library (4 citations) GeCKO sgRNA human library (2 citations)

6 protocols using sgrna library

1

Functional Genomics Screening in EKVX Cells

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We first infect EKVX cells with virus for encoding Cas9, and selected with blasticidin (3513–03-9, Solarbio) for two weeks. Monoclone was picked to generate a stable cell line (EKVX-Cas9). EKVX-Cas9 was further infected with the sgRNA library (addgene, #73178). After 24 h infection, a portion of the cells were collected as a control. The cells were selected with puromycin (1 μg/mL) for a week to eliminate uninfected cells. The infected cells were inoculated at 2 × 106 cells subcutaneously into nude mice. After 14 days, the tumor was harvested to prepare genomic DNA. DNA fragment containing CRISPR sequence were PCR amplified and sent for sequencing.
Liu L., Lei Y., Chen W., Zhou Q., Zheng Z., Zeng G., Liu W., Feng P., Zhang Z., Yu L, & Chen L. (2022). In vivo genome-wide CRISPR screening identifies ZNF24 as a negative NF-κB modulator in lung cancer. Cell & Bioscience, 12, 193.
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2

ATAC-seq Library Preparation with Mitochondrial DNA Depletion

Cited 1 time
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Harvested primary cells were immediately used to prepare ATAC-seq libraries (Buenrostro et al., 2013 (link)). Briefly, isolated cells were washed in PBS and resuspended in buffer containing transposase Tn5 and incubated at 37°C for 30 minutes. Number of cells used to prepare each library has been summarised in Table S1. Tagmented DNA was PCR amplified for 10–16 cycles.
Mitochondrial DNA present in the libraries was cleaved using a CRISPR-Cas9 based method described in (Wu et al., 2016 (link)). sgRNA library targeting mitochondrial DNA was purchased from Addgene (#82480). In vitro transcription was performed to produce the gRNAs using MEGAshortscript Kit (Thermo Fisher Scientific). ATAC-seq libraries were treated with 10 μg Cas9 protein and 15 μg of sgRNA in a 40 μL reaction for 2 hours at 37°C. The libraries were purified using Qiaquick PCR purification kit (Qiagen) and sequenced using Illumina Hi-Seq 2500 (50 bp paired end) or Next-Seq (75 bp paired end).
Murthy P.K., Xi R., Arguijo D., Everitt J.I., Kocak D.D., Kobayashi Y., Bozec A., Vicent S., Ding S., Crawford G.E., Hsu D., Tata P.R., Reddy T, & Shen X. (2022). Epigenetic basis of oncogenic-Kras mediated epithelial-cellular proliferation and plasticity. Developmental cell, 57(3), 310-328.e9.
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3

Lentiviral Transduction of Glioma Stem Cells

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The sgRNA library and single-targeted sgRNA lentiviral plasmids for GSC transduction were purchased from Addgene (#73179 and #52961, respectively). Lentiviral particles were generated as previously described67 (link). For lentiviral transduction, GSC tumorspheres were dissociated into single cells using Accutase (Innovative Cell Technologies), resuspended in GSC media, and optimal lentivirus was added in order to achieve 30–40% infection efficiency. GSCs were then washed once after 12 h, resuspended in fresh GSC media, and cultured for 2 days. Transduced cells were enriched by puromycin (Thermo Fisher Scientific, final concentration 1 μg/ml) selection for seven continuous days.
Bernareggi D., Xie Q., Prager B.C., Yun J., Cruz L.S., Pham T.V., Kim W., Lee X., Coffey M., Zalfa C., Azmoon P., Zhu H., Tamayo P., Rich J.N, & Kaufman D.S. (2022). CHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity. Nature Communications, 13, 1899.
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4

Glioblastoma Stem Cell Lentiviral Transduction

Cited 2 times
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GSCs were acquired from patient specimens at City of Hope under protocols approved by the IRB, and maintained as tumorspheres in GSC media as previously described (4 (link),91 (link)). GSC lines used in this study to test CAR T cell function are IDH1/2-wildtype. The sgRNA library and single-targeted sgRNA lentiviral plasmids (containing a puromycin-resistance gene) for GSC transduction were purchased from Addgene (#73179 and #52961, respectively). Lentiviral particles were generated as previously described (92 (link)). For lentiviral transduction, GSC tumorspheres were dissociated into single cells using Accutase (Innovative Cell Technologies), resuspended in GSC media and lentivirus was added at a 1:50 v/v ratio. GSCs were then washed once after 12 hours, resuspended in fresh GSC media and cultured for 3 days. To ensure that only transduced cells were expanded for further assays, GSCs were selected by puromycin (Thermo Fisher Scientific) for 7 continuous days, with a 1:10000 v/v ratio into GSC media.
Wang D., Prager B.C., Gimple R.C., Aguilar B., Alizadeh D., Tang H., Lv D., Starr R., Brito A., Wu Q., Kim L.J., Qiu Z., Lin P., Lorenzini M.H., Badie B., Forman S.J., Xie Q., Brown C.E, & Rich J.N. (2020). CRISPR Screening of CAR T Cells and Cancer Stem Cells Reveals Critical Dependencies for Cell-Based Therapies. Cancer discovery, 11(5), 1192-1211.
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5

CRISPR Screening for Cisplatin Resistance

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The sgRNA library was obtained from Addgene. HEK 293 T cells were plated on 10-cm dishes to clone the lentiCRISPR vector and the control sgRNA (sgCtrl), following the protocol of GeCKO v2. Next, T24 cells were infected with slow virus at 0.4 functional multiplicity of infection (MOI) and selected by 2 µg/mL puromycin treatment for 48 h after transfection. Subsequently, 2 × 106 transduced T24 cells were treated with 0.25 µM cisplatin or an equal volume of dimethylformamide (DMF) for 7 or 14 days. Finally, the cells were collected for subsequent genomic DNA analyses.
Genomic DNA was isolated using the TIANamp Genomic DNA Kit (Tiangen, China), and amplified by PCR, according to the manufacturer’s protocol. DNA fragments were selected with 2% agarose gels and subjected to HiSeq2500 (Illumina) for sequencing. The data were then analyzed using the MAGeCK software. The candidate genes in the DMF group were defined by a fold change < 2 or > -2 and Bayes facto r < 0. For the cisplatin-treated group, the gene was set at a fold change < -2 and Bayes factor > 2. The KEGG and BioCarta databases were used to determine the gene ontology (GO) and pathway analyses, respectively.
Shi Z.D., Hao L., Han X.X., Wu Z.X., Pang K., Dong Y., Qin J.X., Wang G.Y., Zhang X.M., Xia T., Liang Q., Zhao Y., Li R., Zhang S.Q., Zhang J.H., Chen J.G., Wang G.C., Chen Z.S, & Han C.H. (2022). Targeting HNRNPU to overcome cisplatin resistance in bladder cancer. Molecular Cancer, 21, 37.
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6

Lentivirus Production Protocol

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For lentivirus production, HEK293T cells were seeded into two T225 flasks (Nunc) at a density of 6 × 105 cells/ml (30 ml per flask) and incubated for 24 h, after which a confluence of 80 % is reached. We added 90 μg of sgRNA library, 60 μg psPAX2, and 20 μg of pMDM2 (both from Addgene) to a total of 6 ml RPMI and 300 μl of TransIT (Mirus) was added to 5.7 ml of RMPI. After 10 min, both solutions were mixed and incubated for another 30 min before adding to both flasks (6 ml/flask). After 24 h, medium was exchanged to DMEM containing 10 % fetal calf serum and 1 U/ml DNAseI (Thermo Fisher). Viral supernatant was harvested 48 h after transfection and stored at −80 °C until use. For determining multiplicity of infection (MOI), lentiviral supernatant was generated using the GFP-expressing vector pLKO-G3 (Addgene) under the same conditions and used as a surrogate.
Heigwer F., Zhan T., Breinig M., Winter J., Brügemann D., Leible S, & Boutros M. (2016). CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries. Genome Biology, 17, 55.
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