High-Efficiency CRISPR Screening Libraries

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.

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.

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.

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.