Genome-wide CRISPR screening reveals key genes and pathways associated with 20-hydroxyecdysone signal transduction in the silkworm(Bombyx mori)

Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E).Despite important progress in understanding various aspects of silkworm biology,the hormone signaling pathway in the silkworm remains poorly understood.Genome-wide screening using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-based libraries has recently emerged as a novel method for analyzing genome function,enabling further research into essential genes,drug targets,and virus-host interaction.Previously,we constructed a genome-wide CRISPR/Cas9-based library of the silkworm(Bombyx mori)and successfully revealed the genes involved in biotic or abiotic stress factor responses.In this study,we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action.Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus.Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity,interfere with intracellular nutrition and energy metabolism,and eventually cause cell apoptosis.The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes,which had increased tolerance to 20E.Our findings provide a panoramic overview of signaling in response to 20E in the silkworm,underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.

CRISPR-Cas9-based genome-wide screening identified novel targets for treating sorafenib-resistant hepatocellular carcinoma:a cross-talk between FGF21 and the NRF2 pathway

The treatment of hepatocellular carcinoma(HCC)has been dominated by multikinase inhibitors for more than a decade.However,drug resistance can severely restrict the efficacy of these drugs.Using CRISPR/CAS9 genome library screening,we evaluated Kelch-like ECH-associated protein 1(KEAP1)as a key regulator of sorafenib’s susceptibility in HCC.We also investigated whether KEAP1’s knockdown can stabilize nuclear factor(erythroid-derived 2)-like 2(NRF2)protein levels that led to sorafenib’s resistance,including an NRF2 inhibitor that can synergize with sorafenib to abolish HCC’s growth in vitro and in vivo.Furthermore,we clarified that fibroblast growth factor 21(FGF21)is an important downstream regulator of NRF2 in HCC.Intriguingly,we observed that FGF21 bound to NRF2 through the C-terminus of FGF21,thereby stabilizing NRF2 by reducing its ubiquitination and generating a positive feedback loop in sorafenib-resistant HCC.These findings,therefore,propose that targeting FGF21 is a promising strategy to combat HCC sorafenib’s resistance.

SNX11 Identified as an Essential Host Factor for SFTS Virus Infection by CRISPR Knockout Screening

Severe fever with thrombocytopenia syndrome virus(SFTSV)is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome(SFTS)in humans.The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized.Using a genome-wide CRISPR-based screening strategy,we identified a host cellular protein,sorting nexin 11(SNX11)which is involved in the intracellular endosomal trafficking pathway,as an essential cell factor for SFTSV infection.An SNX11-KO HeLa cell line was established,and SFTSV replication was significantly reduced.The glycoproteins of SFTSV were detected and remained in later endosomal compartments but were not detectable in the endoplasmic reticulum(ER)or Golgi apparatus.pH values in the endosomal compartments of the SNX11-KO cells increased compared with the pH of normal HeLa cells,and lysosomal-associated membrane protein 1(LAMP1)expression was significantly elevated in the SNX11-KO cells.Overall,these results indicated that penetration of SFTSV from the endolysosomes into the cytoplasm of host cells was blocked in the cells lacking SNX11.Our study for the first time provides insight into the important role of the SNX11 as an essential host factor in the intracellular trafficking and penetrating process of SFTSV infection via potential regulation of viral protein sorting,membrane fusion,and other endocytic machinery.

A simple and efficient method for CRISPR/Cas9-induced mutant screening

The clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated protein 9（Cas9） system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction（PCR）/restriction enzyme（RE） assay, T7 endonuclease I（T7EI） assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting（HRM） analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are timeand labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we described a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR（ACT-PCR）, for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.

CRISPR/Cas9 screening identifies a kinetochore-microtubule dependent mechanism for Aurora-A inhibitor resistance in breast cancer

Background:Overexpression of Aurora-A(AURKA)is a feature of breast cancer and associates with adverse prognosis.The selective Aurora-A inhibitor alisertib(MLN8237)has recently demonstrated promising antitumor responses as a single agent in various cancer types but its phase III clinical trial was reported as a failure since MLN8237 did not show an apparent effect in prolonging the survival of patients.Thus,identification of potential targets that could enhance the activity of MLN8237 would provide a rationale for drug combination to achieve better therapeutic outcome.Methods:Here,we conducted a systematic synthetic lethality CRISPR/Cas9 screening of 507 kinases using MLN8237 in breast cancer cells and identified a number of targetable kinases that displayed synthetic lethality interactions with MLN8237.Then,we performed competitive growth assays,colony formation assays,cell viability assays,apoptosis assays,and xenograft murine model to evaluate the synergistic therapeutic effects of Haspin(GSG2)depletion or inhibition with MLN8237.For mechanistic studies,immunofluorescence was used to detect the state of microtubules and the localization of Aurora-B and mitotic centromere-associated kinesin(MCAK).Results:Among the hits,we observed that Haspin depletion or inhibition marginally inhibited breast cancer cell growth but could substantially enhance the killing effects of MLN8237.Mechanistic studies showed that co-treatment with Aurora-A and Haspin inhibitors abolished the recruitment of Aurora-B and mitotic centromere-associated kinesin(MCAK)to centromeres which were associated with excessive microtubule depolymerization,kinetochore-microtubule(KT-MT)attachment failure,and severe mitotic catastrophe.We further showed that the combination of MLN8237 and the Haspin inhibitor CHR-6494 synergistically reduced breast cancer cell viability and significantly inhibited both in vitro and in vivo tumor growth.Conclusions:These findings establish Haspin as a synthetic lethal target and demonstrate CHR-6494 as a potential combinational drug for promoting the therapeutic effects of MLN8237 on breast cancer.

The advancements,challenges,and future implications of the CRISPR/Cas9 system in swine research

Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(CRISPR/Cas9)genome editing technology has dramatically influenced swine research by enabling the production of high-quality disease-resistant pig breeds,thus improving yields.In addition,CRISPR/Cas9 has been used extensively in pigs as one of the tools in biomedical research.In this review,we present the advancements of the CRISPR/Cas9 system in swine research,such as animal breeding,vaccine development,xenotransplantation,and disease modeling.We also highlight the current challenges and some potential applications of the CRISPR/Cas9 technologies.

Genome-wide CRISPR screen identifies synthetic lethality between DOCK1 inhibition and metformin in liver cancer

Metformin is currently a strong candidate anti-tumor agent in multiple cancers.However,its anti-tumor effectiveness varies among different cancers or sub-populations,potentially due to tumor heterogeneity.It thus remains unclear which hepatocellular carcinoma(HCC)patient subpopulation(s)can benefit from met-formin treatment.Here,through a genome-wide CRISPR-Cas9-based knockout screen,we find that DOCK1 levels determine the anti-tumor effects of met-formin and that DOCK1 is a synthetic lethal target of metformin in HCC.Mechanistically,metformin promotes DOCK1 phosphorylation,which activates RAC1 to facilitate cell survival,leading to metformin resistance.The DOCK1-selective inhibitor,TBOPP,potentiates anti-tumor activity by metformin in vitro in liver cancer cell lines and patient-derived HCC organoids,and in vivo in xenografted liver cancer cells and immunocompetent mouse liver cancer models.Notably,metformin improves overall survival of HCC patients with low DOCK1 levels but not among patients with high DOCK1 expression.This study shows that metformin effective-ness depends on DOCK1 levels and that combining metformin with DOCK1 inhibition may provide a promising personalized therapeutic strategy for met-formin-resistant HCC patients.

Key elements for designing and performing a CRISPR/Cas9-based genetic screen

Reverse genetic screens are invaluable for uncovering gene functions, but are traditionally hampered by some technical limitations. Over the past few years, since the advent of the revolutionary CRISPR/Cas9 technology, its power in genome editing has been harnessed to overcome the traditional limitations in reverse genetic screens, with successes in various biological contexts. Here, we outline these CRISPR/Cas9-based screens, provide guidance on the design of effective screens and discuss the potential future directions of development of this field.

CRISPR Screens Identify Essential Cell Growth Mediators in BRAF Inhibitor-resistant Melanoma

BRAF is a serine/threonine kinase that harbors activating mutations in^7%of human malignancies and^60%of melanomas.Despite initial clinical responses to BRAF inhibitors,patients frequently develop drug resistance.To identify candidate therapeutic targets for BRAF inhibitor resistant melanoma,we conduct CRISPR screens in melanoma cells harboring an activating BRAF mutation that had also acquired resistance to BRAF inhibitors.To investigate the mechanisms and pathways enabling resistance to BRAF inhibitors in melanomas,we integrate expression,ATAC-seq,and CRISPR screen data.We identify the JUN family transcription factors and the ETS family transcription factor ETV5 as key regulators of CDK6,which together enable resistance to BRAF inhibitors in melanoma cells.Our findings reveal genes contributing to resistance to a selective BRAF inhibitor PLX4720,providing new insights into gene regulation in BRAF inhibitor resistant melanoma cells.

Bioinformatics approaches to analyzing CRISPR screen data:from dropout screens to single-cell CRISPR screens

Background:Pooled CRISPR screen is a promising tool in drug targets or essential genes identification with the utilization of three different systems including CRISPR knockout(CRISPRko),CRISPR interference(CRISPRi)and CRISPR activation(CRISPRa).Aside from continuous improvements in technology,more and more bioinformatics methods have been developed to analyze the data obtained by CRISPR screens which facilitate better understanding of physiological effects.Results:Here,we provide an overview on the application of CRISPR screens and bioinformatics approaches to analyzing different types of CRISPR screen data.We also discuss mechanisms and underlying challenges for the analysis of dropout screens,sorting-based screens and single-cell screens.Conclusion:Different analysis approaches should be chosen based on the design of screens.This review will help community to better design novel algorithms and provide suggestions for wet-lab researchers to choose from different analysis methods.

SeqCor:correct the effect of guide RNA sequences in clustered regularly interspaced short palindromic repeats/Cas9 screening by machine learning algorithm

Clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9-based screening using various guide RNA(g RNA)libraries has been executed to identify functional components for a wide range of phenotypes with regard to numerous cell types and organisms.Using data from public CRISPR/Cas9-based screening experiments,we found that the sequences of g RNAs in the library influence CRISPR/Cas9-based screening.As building a standard strategy for correcting results of all g RNA libraries is impractical,we developed Seq Cor,an open-source programming bundle that enables researchers to address the result bias potentially triggered by the composition of g RNA sequences via the organization of g RNA in the library used in CRISPR/Cas9-based screening.Furthermore,Seq Cor completely computerizes the extraction of sequence features that may influence single-guide RNA knockout efficiency using a machine learning approach.Taken together,we have developed a software program bundle that ought to be beneficial to the CRISPR/Cas9-based screening platform.

Low-density lipoprotein receptor-related protein 1 is a CROPs-associated receptor for Clostridioides infection toxin B

As the leading cause of worldwide hospital-acquired infection,Clostridioides difficile(C.difficile)infection has caused heavy economic and hospitalized burden,while its pathogenesis is not fully understood.Toxin B(Tcd B)is one of the major virulent factors of C.difficile.Recently,CSPG4 and FZD2 were reported to be the receptors that mediate Tcd B cellular entry.However,genetic ablation of genes encoding these receptors failed to completely block Tcd B entry,implicating the existence of alternative receptor(s)for this toxin.Here,by employing the CRISPR-Cas9 screen in CSPG4-deficient He La cells,we identified LDL receptor-related protein-1(LRP1)as a novel receptor for Tcd B.Knockout of LRP1 in both CSPG4-deficient He La cells and colonic epithelium Caco2 cells conferred cells with increased Tcd B resistance,while LRP1 overexpression sensitized cells to Tcd B at a low concentration.Co-immunoprecipitation assay showed that LRP1 interacts with full-length Tcd B.Moreover,CROPs domain,which is dispensable for Tcd B’s interaction with CSPG4 and FZD2,is sufficient for binding to LRP1.As such,our study provided evidence for a novel mechanism of Tcd B entry and suggested potential therapeutic targets for treating C.difficile infection.