CRISPR library 101 (Part I)
what are the popular applications of CRISPR library?
The CRISPR library is the most popular high-throughput screening tool. In this article, Ubigene will give you a brief introduction of this high-throughput screening method from two aspects: what the CRISPR library is and what its applications are. Hope this can provide you with some research ideas.
CRISPR library introduction
CRISPR library is a high-throughput gene screening method based on CRISPR/Cas9 technology. Construct a plasmid library by high-throughput synthesis of sgRNA, package it as lentivirus, and then infect the cells with low MOI (usually < 0.3), to ensure that only one sgRNA enters a cell, and then screen the cells (such as drug treatment, hypoxia treatment, virus infection or the self viability of the cells), collect the screened cells for NGS sequencing, and analyze the enrichment or depletion of sgRNA between the experimental group and the control group, then obtain host factors related to the screening condition.
Compared with cDNA library and RNAi library, CRISPR/Cas9 has the advantages of versatility, low noise, high knockout efficiency, and less off-target effect, and is the preferred method for large-scale gene function screening.
CRISPR library applications
In addition to the unique advantages mentioned above, the broad application prospect also contributes to the popularity of CRISPR library. Here are a few common applications.
1. Cancer treatment
1）Identify essential genes of tumor and target oncogenes for treatment
Malignant tumor accumulates many gene mutations, but the maintenance of malignant phenotype of these tumor cells usually depends on only one or some of the oncogenes activated by mutations. This phenomenon is called oncogene addiction, and these oncogenes are also called driver oncogenes. Blocking the activity of these driver oncogenes can induce tumor cells to rapid apoptosis, growth arrest or differentiation, but with little impact on normal cells. This difference brings hope for targeted therapy of tumors.
In recent years, the extensive application of second-generation sequencing has identified many mutation loci in tumor cells, but it is difficult to distinguish which are driver mutations. It is a simpler and more direct method to find out the driving oncogenes that tumor cells depend on for survival by gene inactivation. CRISPR library can simultaneously target different genes throughout the genome, which is an excellent tool to achieve high-throughput gene inactivation. In 2014, Shalem et al.  first identified the key genes for the survival of human melanoma cells and pluripotent stem cells using the GeCKO Library (Fig. 1). Wang et al.  identified NCAPG as an essential oncogene for hepatocellular carcinoma tumor growth by CRISPR whole genome library screening. Kiessling et al.  verified the driving genes EGFR of HCC-827 cell line and NRAS and MEK1 of CHP-212 cell line by using CRISPR whole genome library screening, and found new driving genes TBK1 and TRIB2 for further research.
Fig. 1 Screening A375 and HUES62 survival essential genes by CRISPR library
2）Screen synthetic lethality genes to achieve precise treatment
For oncogenes, inhibitors can be used for targeted therapy, but for tumor suppressor genes, the inactivation of their functions makes it impossible to directly target them. So the synthetic lethality gene of the suppressor gene is usually targeted and inactivated. Synthetic lethality refers to the phenomenon that two non-lethal mutant genes inactivate at the same time leading to cell death. If there is inactivation of gene A in tumor cells, drugs can be used to inhibit its synthetic lethality gene B. By inactivate both genes in tumor cells, cell death would occur. However, healthy somatic cells can still have normal physiological functions because they have normal gene A. Thus, the drug specifically kills tumor cells (see Fig. 2) .
Fig.2 Synthetic lethality therapy
The most typical application of synthetic lethality is the treatment of BRCA1/2 mutated tumors with PARP inhibitors. By 2020, the global sales of PARP inhibitors had exceeded 2 billion US dollars. So how to find more gene pairs with synthetic lethality? CRISPR library high-throughput screening plays a big role! CRISPR library screening for synthetic lethality generally has two logics. One is to directly design a dual-gRNA vector, that is, to simultaneously target two genes in one cell and screen gene pairs with synthetic lethality. Shen et al.  used this method to screen 73 genes in three cancer cell lines (Hela, A549 and 293T) by 150000 combinations and found 120 gene pairs with synthetic lethality (see Fig. 3). Another logic is to screen a library on a single tumor suppressor gene knockout cell line to screen out targets that have synthetic lethal effects with the knockout gene. Feng et al. used this method to first construct 12 known tumor suppressor gene knockout cell lines on 293A, and then screen wild-type and knockout cell lines with a genome-wide knockout library to analyze the changes in gRNA abundance before and after cell screening. Potential synthetic lethal genes were identified by comparing the abundance differences of gRNAs between wild-type and knockout cell lines after CRISPR library screening.
Fig. 3 Screening gene interaction relationships using CRISPR library
3）Search for tumor drug resistance related genes, study drug resistance mechanisms, and optimize treatment strategies
The main problem of tumor molecular targeted drugs is drug resistance, so it is of great clinical significance to explore the mechanism of drug resistance. Through positive screening of CRISPR library, most cells died due to drug sensitivity, and some cells survived due to drug resistance by knockout specific gene. The genes related to drug resistance can be screened out by detecting the gRNAs carried by the surviving cells.
Vemurafenib is approved for the treatment of advanced melanoma carrying the BRAFV600E mutation. In order to study the drug resistance mechanism of Vemurafenib, Shalem et al.  designed a knockout library containing 64751 sgRNAs and targeting 18080 genes in the whole genome. After cell infection and expansion, nf1, med12, nf2, cul3, tada2b, tada1 and other drug resistance related genes were screened out. A new hypothesis of the mechanism of Vemurafenib tumor resistance was proposed.
2. Screening host factors related to viral infection
CRISPR library screening is often used to study host factors related to viral infection. For hepatitis C virus, dengue virus, Japanese encephalitis virus and West Nile virus, a number of host factors related to endoplasmic reticulum function and receptors have been screened out. For example, Zhang et al.  verified nine human genes required for flavivirus infection through genome-wide screening of CRISPR/Cas9, all of which are related to endoplasmic reticulum (ER) functions (including translocation, protein degradation and N-linked glycosylation). Ma et al.  screened the whole genome by designing a library containing 77406 sgRNAs and targeting 20121 genes, and then obtained seven genes belong to the endoplasmic reticulum related protein degradation pathway (ERAD), such as EMC2, EMC3 and SEL1L. Further experiments showed that knockdown of these genes would not prevent the replication of West Nile virus (WNV), but would prevent the virus from killing cells.
3. Screening of antibody target
It is quite simple to use peptides or purified proteins to verify antibodies in immune experiments, but it is relatively difficult to use the whole cell or other complex antigens to verify antibodies. If the reactivity of the antibody is not detected in Western blotting and immunoprecipitation, it is necessary to apply a variety of techniques at the gene level to determine the antigen specificity of the mAb. The application of CRISPR library can reduce many operation steps and simplify the experiment. 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.  used MT2 cells (T cells chronically infected with human T-cell lymphotropic virus type I HTLV-1) as immunogens 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 on CEM T and Raji/CD4 B cells to sort out cells that do 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 sgRNA targeted CD82. BF4 was confirmed to be a specific antibody against CD82.
Fig. 4 Verification of BF4 antibody specificity using CRISPR library
4. Study of signal pathway
Pyroptosis is an immune defense reaction 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 lipopolysaccharides, can cause cell pyroptosis, but the mechanism remains unknown. Shi et al.  first established a lipopolysaccharide (LPS) electrotransformation method that can induce more than 90% cell pyroptosis, and then transduced a CRISPR knockout library into Tlr4–/– bone marrow-derived immortalized macrophages (iBMDMs) that can normally respond to lipopolysaccharide stimulation, and sequenced and analyzed the cells that survived after lipopolysaccharide induced cell pyroptosis. The analysis results showed that among the five sgRNAs targeting the gasdermin D (GSDMD) gene, four had copy numbers in the top 30, of which two were in the top 10. The subsequent results also further proved that the N-terminal of GSDMD could induce cell pyroptosis. To sum up, the researchers used CRISPR gene library to carry out genome-wide genetic screening, successfully screened the gene GSDMD that can inhibit cell pyroptosis after knockout, and clarified the molecular mechanism of GSDMD as an inflammatory caspase substrate protein that can trigger cell pyroptosis after being cleaved.
Fig. 6 siRNA knockdown validation of screen hits
If you are interested in CRISPR libraries, please pay attention to our next article. We will sort out the types and selection strategies of CRISPR libraries, and help you quickly select CRISPR libraries that meet your experimental requirement!
Ubigene has 8 off-shelf CRISPR libraries, covering the whole genome (human/mouse), kinase, nuclear protein, and metabolic genes. We also provide one-stop services from plasmid library construction, virus packaging, cell screening and NGS sequencing analysis. There are also three types of library systems, CRISPR-KO, CRISPRi and CRISPRa, to meet different needs. Contact us for more products and services details>>
 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.
 Wang Y, Gao B, Tan PY, Handoko YA, Sekar K, Deivasigamani A, Seshachalam VP, OuYang HY, Shi M, Xie C, Goh BKP, Ooi LL, Man Hui K. Genome-wide CRISPR knockout screens identify NCAPG as an essential oncogene for hepatocellular carcinoma tumor growth. FASEB J. 2019 Aug;33(8):8759-8770. doi: 10.1096/fj.201802213RR. Epub 2019 Apr 25. PMID: 31022357; PMCID: PMC6662966.
 Kiessling M K, Schuierer S, Stertz S, et al. Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening[J]. BMC genomics, 2016, 17(1): 1-16.
 Sasaki M, Ogiwara H. Synthetic lethal therapy based on targeting the vulnerability of SWI/SNF chromatin remodeling complex‐deficient cancers[J]. Cancer Science, 2020, 111(3): 774-782.
 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.
 Feng X, Tang M, Dede M, et al. Genome-wide CRISPR screens using isogenic cells reveal vulnerabilities conferred by loss of tumor suppressors[J]. Science advances, 2022, 8(19): eabm6638.
 Zhang R, Miner JJ, Gorman MJ, Rausch K, Ramage H, White JP, Zuiani A, Zhang P, Fernandez E, Zhang Q, Dowd KA, Pierson TC, Cherry S, Diamond MS. A CRISPR screen defines a signal peptide processing pathway required by flaviviruses. Nature. 2016 Jul 7;535(7610):164-8. doi: 10.1038/nature18625. Epub 2016 Jun 17. PMID: 27383988; PMCID: PMC4945490.
 Ma H, Dang Y, Wu Y, Jia G, Anaya E, Zhang J, Abraham S, Choi JG, Shi G, Qi L, Manjunath N, Wu H. A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death. Cell Rep. 2015 Jul 28;12(4):673-83. doi: 10.1016/j.celrep.2015.06.049. Epub 2015 Jul 16. PMID: 26190106; PMCID: PMC4559080.
 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.
 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.