CRISPR library

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CRISPR libraryCRISPR library

CRISPR library is a high-throughput gene screening method established based on CRISPR/Cas9 technology. It identifies phenotype-related genes or screens new drug targets through functional screening, enrichment and deep sequencing analysis. The scope of screening can be the whole genome, a certain gene family, or a certain signal pathway gene. CRISPR library screening has become the preferred platform for large-scale gene function screening benefited from the characteristics of CRISPR/Cas9 such as versatility, low noise, high knockout efficiency, and less off-target effect.

Ubigene focuses on the field of gene editing and has rich experience in cell gene editing. It can provide 35+ off-shelf libraries and one-stop services from high-throughput sgRNA library construction, virus packaging, cell infection, drug screening, NGS sequencing and data analysis, etc.

Libraries in Stock

Ubigene provides human/mouse CRISPR knockout gRNA libraries for whole genome and sub-libraries based on kinases, cell cycle, membrane proteins, metabolic genes, etc., to help you quickly screen genes related to phenotypes or trigger specific functions of cells, and tap potential targets, such as genes that cause drug resistance or sensitivity of cells, genes that affect the sensitivity of environmental toxins, new genes composed of cellular pathways, and genes that cause specific disease states.

Genome-wide Library
Human
  • EZ-editor™ Human GeCKO v2 genome-wide library Single-plasmid System A/B EZ-editor™ Human GeCKO v2 genome-wide library
  • EZ-editor™ Human GeCKO v2 genome-wide library Dual-plasmid System A/B EZ-editor™ Human GeCKO v2 genome-wide library
  • EZ-editor™ Human Genome-wide Inhibition Library Human CRISPR Inhibition Pooled Library (Dolcetto)
    (#1000000114)
  • EZ-editor™ Human Genome-wide Activation Library EZ-editor™ Human SAM genome-wide library (3-plasmid system) (#1000000074) (Puromycin)
  • EZ-editor™ Human Genome-wide Knockout Library (Brunello) EZ-editor™ Human CRISPR Knockout Pooled Library (Brunello)
    (#73179)
Mouse
  • EZ-editor™ Mouse Genome-wide Activation Library EZ-editor™ Mouse CRISPR Activation Library (SAM - 3 plasmid system)
    (#1000000075)
  • EZ-editor™ Mouse Genome-wide Knockout Library Single-plasmid System A/B EZ-editor™ Mouse GeCKO v2 genome-wide library
Green Monkey
  • EZ-editor™ Green Monkey Genome-wide Knockout Library EZ-editor™ Green monkey sgRNA library†
    (#178284)
Pig
  • Pig sgRNA Knockout Pooled Library Pig CRISPR Knockout Pooled Library
Sub-library
Human
  • EZ-editor™ Human Kinases Knockout Library EZ-editor™ Human Lentiviral sgRNA Library - Kinases
    (#51044)
  • EZ-editor™ Human Nuclear Protein Knockout Library EZ-editor™ Human Lentiviral sgRNA Library - Nuclear
    (#51047)
  • EZ-editor™ Human Metabolic Gene Knockout Library EZ-editor™ Human CRISPR Metabolic Gene Knockout Library
    (#110066)
  • EZ-editor™ Human Membrane Protein Knockout Library EZ-editor™ Membrane protein gRNA library
    (#113345)
  • EZ-editor™ Human Epigenetic Knockout Library EZ-editor™ Human Epigenetic Knockout Library
    (#162256)
  • EZ-editor™ Human Cell-cycle Protein Knockout Library EZ-editor™ Human Lentiviral sgRNA Library - Cell Cycle
    (#51046)
  • EZ-editor™ Human Drug Targets, Kinases, Phosphatases Knockout Library
  • EZ-editor™ Human Apoptosis and Cancer Gene Knockout Library Human CRISPR Deletion Library - Apoptosis and cancer†
    (#101926)
  • EZ-editor™ Human RNA-Binding Protein Knockout Library Yeo Lab RNA-Binding Protein Pooled CRISPR Knockout Library
    (#141438)
  • EZ-editor™ Human CRISPR Knockout Library - Gene expression EZ-editor™ Human CRISPR Deletion Library - Gene expression†(#101928)
  • EZ-editor™ Human CRISPR Knockout Library - Proteostasis EZ-editor™ Human CRISPR Deletion Library - Proteostasis†(#101930)
  • EZ-editor™ Human CRISPR Knockout Library - Trafficking, mitochondrial, motility EZ-editor™ Human CRISPR Deletion Library - Trafficking, mitochondrial, motility†(#101931)
  • EZ-editor™ Human CRISPR Knockout Library - Transcription Factor-DNA Binding Domain EZ-editor™ Human DNA Binding Domain-Focused CRISPR Knockout Library(#123334)
  • EZ-editor™ Human Interferon-Stimulated Gene Knockout Library EZ-editor™ Human Interferon-Stimulated Gene CRISPR Knockout Library(#125753)
  • EZ-editor™ Human CRISPR Knockout Library - lncRNA Splicing-targeting EZ-editor™ Human lncRNA Splicing-targeting CRISPR Library(#119977)
  • EZ-editor™ Human CRISPR Knockout Library - Ubiquitination and de-ubiquitination complex genes (UBDUB) EZ-editor™ Human UBDUB CRISPR Knockout Library(#171531)
  • EZ-editor™ Human miRNA CRISPR Knockout Library EZ-editor™ Lin Human miRNA CRISPR Knockout Library(#112200)
  • EZ-editor™ Human Membrane Protein Activation Library EZ-editor™ Human CRISPR Deletion Library - Membrane proteins†(#101929)
  • EZ-editor™ Human Transcription Factor Knockout Library EZ-editor™ Human Transcription Factor Knockout Library(#162275)
  • EZ-editor™ Human Ubiquitination-Related Protein Knockout Library EZ-editor™ Bonifacino Lab Human ubiquitination-related proteins CRISPR KO library(#174592)
Mouse
  • EZ-editor™ Mouse Metabolic Gene Knockout Library EZ-editor™ Mouse CRISPR Metabolic Gene Knockout Library
    (#160129)
  • EZ-editor™ Mouse Membrane Protein Activation Library EZ-editor™ Mouse Subpooled CRISPRa-v2 Libraries-Membrane Proteins
    (#84003)
  • EZ-editor™ Mouse Membrane Protein CRISPR Interference Library EZ-editor™ Mouse Subpooled CRISPRi-v2 Libraries-Membrane Proteins
    (#83994)
Deliverable
Plasmid / Virus (multiple sizes available)
QC
Coverage rate > 99%, uniformity < 10
Price & More Details

Library Customization

Ubigene provides library customization for CRISPR-KO, CRISPRa, and CRISPRi, including genome-wide libraries and sub-libraries, assisting in high-throughput target screening.

Library Customization
Library Type
CRISPR-KO Library / CRISPRa Library / CRISPRi Library
QC
Coverage rate > 99%, Uniformity < 10
Deliverables
Virus particles (Titer: ≥1*10^8TU/mL) / Plasmid (100μg), NGS Report
Price & More Details
CRISPR-KO library
Cas9:gRNA complex
Cas9 and gRNA in CRISPR library
The gRNAs of this kind of library mainly target the 5' end exons of coding genes. DNA double strand breaks are caused by Cas9 protein cleavage, and knockout is achieved by introducing frameshift mutations through the non homologous end joining (NHEJ) DNA repair mechanism.
CRISPRa library
dCas9-SAM system
dCas9-SAM for CRISPRa library
This kind of library can target the transcription-regulatory regions of coding or non-coding genes, achieve the co-expression of Cas9 protein and MS2-P65-HSF1 activation auxiliary protein by connecting dCas9 protein and transcription activator VP64, effectively upregulate the expression level of the genes, and complete high-throughput gain-of-function screening.
CRISPRi library
dCas9-KRAB
dCas9-KRAB for CRISPRi library
The gRNAs of this kind of library mainly target non-coding genes and essential genes (knockout lethality). Gene knockdown was achieved by co-transfecting the gRNA targeting the upstream regulatory region of the gene and a protein of the catalytically inactive dCas9 fused with the transcription repressor KRAB (Kruppel-related frame domain).
CRISPR-KO library
Cas9 and gRNA in CRISPR library
The gRNAs of this kind of library mainly target the 5' end exons of coding genes. DNA double strand breaks are caused by Cas9 protein cleavage, and knockout is achieved by introducing frameshift mutations through the non homologous end joining (NHEJ) DNA repair mechanism.
CRISPRa library
dCas9-SAM for CRISPRa library
This kind of library can target the transcription-regulatory regions of coding or non-coding genes, achieve the co-expression of Cas9 protein and MS2-P65-HSF1 activation auxiliary protein by connecting dCas9 protein and transcription activator VP64, effectively upregulate the expression level of the genes, and complete high-throughput gain-of-function screening.
CRISPRi library
dCas9-KRAB for CRISPRi library
The gRNAs of this kind of library mainly target non-coding genes and essential genes (knockout lethality). Gene knockdown was achieved by co-transfecting the gRNA targeting the upstream regulatory region of the gene and a protein of the catalytically inactive dCas9 fused with the transcription repressor KRAB (Kruppel-related frame domain).

CRISPR Screening

Ubigene provides one-stop customized screening services including sgRNA library construction, virus packaging, cell screening, NGS sequencing, and data analysis, etc, covering research directions such as tumor-related genes, virus infection targets, signal pathway regulation, metabolic research, and immune research. The coverage of the genome-wide pooled library is up to 99%, helping you efficiently screen targets!

CRISPR Screening
Screen Type
CRISPR-KO Screening / CRISPRa Screening / CRISPRi Screening
Deliverables
CRISPR Screen Sequencing & Analysis Report
Price & More Details

CRISPR screening workflow

CRISPR library Technical Process
CRISPR library Technical Process
Screening functional genes of tumor
Negative screening of gene pairs with synthetic lethality

Synthetic lethality refers to the phenomenon that when two non-lethal mutant genes occur alone, they will not cause cell death, but when they occur simultaneously, they will cause cell death, which is one of the new research directions in the field of antitumor drugs. Because tumor cells often carry many point mutations, how to specifically kill tumor cells with high mutation rate without affecting normal cells is a major pursuit of antitumor drug research and development. Starting from the idea of synthetic lethality, Shen et al [2] designed a dual-gRNA library to screen the synthetic lethal interaction network. Different from the general sgRNA library, each vector in the dual-gRNA library contains two gRNAs, one targeting common mutated tumor suppressor genes in tumors and the other targeting genes that can be perturbed by anticancer drugs. They used this system to screen 73 genes in three experimental cancer cell lines (human cervical cancer HeLa, lung cancer A549 and embryonic renal cell carcinoma 293T), with a total of about 150000 gene combinations. By detecting gRNA abundance changes at different time points, they further analyzed and screened 120 synthetic lethal interactions, providing new targets for the development of new cancer drugs.

Viral infection related study
Screening of HIV therapeutic targets

AIDS caused by HIV infection is a serious threat to human life and health. It is of great significance to clarify the molecular mechanism of HIV breaking through the host cell defense system and develop new targets for HIV treatment. Park et al. [3] infected cas9 virus on CCRF-CEM cells stably expressing CCR5 hygR and HIV-1 LTR-GFP, and screened a clone (GXRCas9 cell) that highly expressed CCR5 before HIV infection and low expressed EGFP but high expressed EGFP after infection as a tool cell for screening HIV infection targets. Specifically, GXRCas9 cells were infected with a lentiviral library containing 187536 sgRNAs (targeting 18543 genes), and these T cells lacking different receptors were infected with the HIV virus strain JR-CSF. Then, GFP negative and positive T cells were sorted out by flow cytometry, and the GFP negative cell population and the uninfected HIV virus cell population were sequenced to analyze the difference in sgRNA abundance between the two groups of cells. Finally, five genes with the largest change in sgRNA abundance were screened out. Among them, CD4 and CCR5 are the receptors of HIV-infected T cells. TPST2 and SLC35B2 modify CCR5 to facilitate the binding of HIV, while the gene encoding leukocyte adhesion factor ALCAM is related to the transmission of HIV between cells. The five genes screened do not affect the survival of T cells after being knocked out, but can make T cells resist HIV infection. Therefore, these five genes can be used as potential targets for HIV treatment, providing new ideas and ways for the prevention and treatment of AIDS.

Antibody target screening
Recognition of monoclonal antibody specific target antigens and their epitopes

It is quite simple to use peptides or purified proteins to verify antibodies in immune experiments, but it is relatively difficult to use whole cells or other complex antigens to verify antibodies. If no antibody reactivity is detected in Western blotting and immunoprecipitation, a variety of gene manipulation level techniques need to be applied to determine the antigen specificity of mAbs. BF4 is an antibody that can bind to the viral biofilm on the surface of uninfected lymphocytes, neutrophils and HTLV-1 infected cells. Zotova et al [4] used MT2 cells (human T cell lymphotropic virus type I HTLV-1 chronically infecting T cells) as immunogen to trigger mouse immunity and obtained a HTLV-1 biofilm specific monoclonal antibody BF4. Based on the idea of screening BF4 antigen knockout cells by transducing CRISPR knockout library into BF4 positive cells, they transduced GeCKO library to CEM T and Raji/CD4 B cells to sort out cells that did not bind to BF4. After two rounds of repeated sorting, the proportion of negative cells reached more than 99%. Researchers sequenced these cells and found that about 80% of sgRNAs targeted CD82. BF4 was confirmed to be a specific antibody against CD82.

Signal pathway
Studying the mechanism of pyroptosis

Pyroptosis is an immune defense response initiated by the body after sensing the infection of pathogenic microorganisms. Inflammation-activated caspase-1 and caspase-4, Caspase-5 and caspase-11, which recognize bacterial lipopolysaccharide, can cause pyroptosis, but the mechanism remains unknown. Shi et al [5] first established a lipopolysaccharide (LPS) electroporation method that can induce pyroptosis in more than 90% of cells, and then transduced the CRISPR knockout library into Tlr4-/- iBMDMs that can normally respond to lipopolysaccharide stimulation, and sequenced the cells that survived pyroptosis induced by lipopolysaccharide. The analysis results showed that four of the five sgRNAs targeting gasdermin D (GSDMD) gene had the top 30 copy numbers, and two of them were in the top 10 positions. The subsequent results also further proved that the N-terminus of GSDMD could induce pyroptosis. In summary, researchers used CRISPR library to conduct genome-wide genetic screening, successfully screened the gene GSDMD that can inhibit pyroptosis after knockout, and clarified the molecular mechanism of GSDMD as an inflammatory caspase substrate protein that can trigger pyroptosis after being cleaved.

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References
[1]Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014 Jan 3;343(6166):84-87. doi: 10.1126/science.1247005. Epub 2013 Dec 12. PMID: 24336571; PMCID: PMC4089965.
[2]Shen JP, Zhao D, Sasik R, Luebeck J, Birmingham A, Bojorquez-Gomez A, Licon K, Klepper K, Pekin D, Beckett AN, Sanchez KS, Thomas A, Kuo CC, Du D, Roguev A, Lewis NE, Chang AN, Kreisberg JF, Krogan N, Qi L, Ideker T, Mali P. Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions. Nat Methods. 2017 Jun;14(6):573-576. doi: 10.1038/nmeth.4225. Epub 2017 Mar 20. PMID: 28319113; PMCID: PMC5449203.
[3]Park RJ, Wang T, Koundakjian D, Hultquist JF, Lamothe-Molina P, Monel B, Schumann K, Yu H, Krupzcak KM, Garcia-Beltran W, Piechocka-Trocha A, Krogan NJ, Marson A, Sabatini DM, Lander ES, Hacohen N, Walker BD. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nat Genet. 2017 Feb;49(2):193-203. doi: 10.1038/ng.3741. Epub 2016 Dec 19. PMID: 27992415; PMCID: PMC5511375.
[4]Zotova A, Zotov I, Filatov A, Mazurov D. Determining antigen specificity of a monoclonal antibody using genome-scale CRISPR-Cas9 knockout library. J Immunol Methods. 2016 Dec;439:8-14. doi: 10.1016/j.jim.2016.09.006. Epub 2016 Sep 21. PMID: 27664857.
[5]Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, Zhuang Y, Cai T, Wang F, Shao F. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature. 2015 Oct 29;526(7575):660-5. doi: 10.1038/nature15514. Epub 2015 Sep 16. PMID: 26375003.

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