Plant Functional Genomics Based on High-Throughput CRISPR Library Knockout Screening: A Perspective.

Plant biology studies in the post-genome era have been focused on annotating genome sequences' functions. The established plant mutant collections have greatly accelerated functional genomics research in the past few decades. However, most plant genome sequences' roles and the underlying regulatory networks remain substantially unknown. Clustered, regularly interspaced short palindromic repeat (CRISPR)-associated systems are robust, versatile tools for manipulating plant genomes with various targeted DNA perturbations, providing an excellent opportunity for high-throughput interrogation of DNA elements' roles. This study compares methods frequently used for plant functional genomics and then discusses different DNA multi-targeted strategies to overcome gene redundancy using the CRISPR-Cas9 system. Next, this work summarizes recent reports using CRISPR libraries for high-throughput gene knockout and function discoveries in plants. Finally, this work envisions the future perspective of optimizing and leveraging CRISPR library screening in plant genomes' other uncharacterized DNA sequences.

Uncovering the bookshelves of CRISPR-based libraries: Advances and applications in cancer studies.

The advent of CRISPR/Cas9 technology has revolutionized the genome editing field. CRISPR-based libraries have become powerful tools for high-throughput functional genomics and genetic screening. CRISPR-based libraries can represent a powerful approach to uncovering genes related to chemoresistance and therapy efficacy and to studying cancer cells' fitness. In this review, we conducted an extensive literature search and summarized multiple studies that utilized these libraries in both in vitro and in vivo research, emphasizing their key findings. We provide an overview of the design, construction, and applications of CRISPR-based libraries in different cancer-focused studies and discuss the different types of CRISPR-based libraries. We finally point out the challenges associated with library design, including guide RNA selection, off-target effects, and library complexity. This review provides an overview of the work conducted with CRISPR libraries in the search for new targets that could potentially assist in cancer therapy by contributing to functional approaches.

Nanopore sequencing improves construction of customized CRISPR-based gene activation libraries.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based screening has emerged as a powerful tool for identifying new gene targets for desired cellular phenotypes. The construction of guide RNA (gRNA) pools largely determines library quality and is usually performed using Golden Gate assembly or Gibson assembly. To date, library construction methods have not been systematically compared, and the quality check of each batch has been slow. In this study, an in-house nanopore sequencing workflow was established for assessing the current methods of gRNA pool construction. The bias of pool construction was reduced by employing the polymerase-mediated non-amplifying method. Then, a small gRNA pool was utilized to characterize stronger activation domains, specifically MED2 (a subunit of mediator complex) and HAP4 (a heme activator protein), as well as to identify better gRNA choices for dCas12a-based gene activation in Saccharomyces cerevisiae. Furthermore, based on the better CRISPRa tool identified in this study, a custom gRNA pool, which consisted of 99 gRNAs targeting central metabolic pathways, was designed and employed to screen for gene targets that could improve ethanol utilization in S. cerevisiae. The nanopore sequencing-based workflow demonstrated here should provide a cost-effective approach for assessing the quality of customized gRNA library, leading to faster and more efficient genetic and metabolic engineering in S. cerevisiae.

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.

A CRISPR activation screen identifies MUC-21 as critical for resistance to NK and T cell-mediated cytotoxicity.

BACKGROUND: Immunotherapy has significantly advanced cancer treatments, but many patients do not respond to it, partly due to immunosuppressive mechanisms used by tumor cells. These cells employ immunosuppressive ligands to evade detection and elimination by the immune system. Therefore, the discovery and characterization of novel immunosuppressive ligands that facilitate immune evasion are crucial for developing more potent anti-cancer therapies. METHODS: We conducted gain-of-function screens using a CRISPRa (CRISPR activation) library that covered the entire human transmembrane sub-genome to identify surface molecules capable of hindering NK-mediated cytotoxicity. The immunosuppressive role and mechanism of MUC21 were validated using NK and T cell mediated cytotoxicity assays. Bioinformatics tools were employed to assess the clinical implications of mucin-21 (MUC21) in cancer cell immunity. RESULTS: Our genetic screens revealed that MUC21 expression on cancer cell surfaces inhibits both the cytotoxic activity of NK cells and antibody-dependent cellular cytotoxicity, but not affecting complement-dependent cytotoxicity. Additionally, MUC21 expression hinders T cell activation by impeding antigen recognition, thereby diminishing the effectiveness of the immune checkpoint inhibitor, anti-PD-L1. Moreover, MUC21 expression suppress the antitumor function of both CAR-T cells and CAR-NK cells. Mechanistically, MUC21 facilitates immune evasion by creating steric hindrance, preventing interactions between cancer and immune cells. Bioinformatics analysis revealed elevated MUC21 expression in lung cancer, which correlated with reduced infiltration and activation of cytotoxic immune cells. Intriguingly, MUC21 expression was higher in non-small cell lung cancer (NSCLC) tumors that were non-responsive to anti-PD-(L)1 treatment compared to responsive tumors. CONCLUSIONS: These findings indicate that surface MUC21 serves as a potent immunosuppressive ligand, shielding cancer cells from NK and CD8+T cell attacks. This suggests that inhibiting MUC21 could be a promising strategy to improve cancer immunotherapy.

CRISPR library screening to develop HEK293-derived cell lines with improved lentiviral vector titers.

Lentiviral (LV) vectors have emerged as powerful tools for treating genetic and acquired human diseases. As clinical studies and commercial demands have progressed, there has been a growing need for large amounts of purified LV vectors. To help meet this demand, we developed CRISPR library screening methods to identify genetic perturbations in human embryonic kidney 293 (HEK293) cells and their derivatives that may increase LV vector titers. Briefly, LV vector-based Human CRISPR Activation and Knockout libraries (Calabrese and Brunello) were used to modify HEK293 and HEK293T cells. These cell populations were then expanded, and integrated LV vector genomes were rescued by transfection. LV vectors were harvested, and the process of sequential transduction and rescue-transfection was iterated. Through this workflow, guide RNAs (gRNAs) that target genes that may suppress or enhance LV vector production were enriched and identified with Next-Generation Sequencing (NGS). Though more work is needed to test genes identified in this screen, we expect that perturbations of genes we identified here, such as TTLL12, which is an inhibitor of antiviral innate immunity may be introduced and multiplexed to yield cell lines with improved LV vector productivity.

In Vivo Genome-Wide CRISPR Activation Screening Identifies Functionally Important Long Noncoding RNAs in Hepatocellular Carcinoma.

BACKGROUND & AIMS: Long noncoding RNAs (lncRNAs) are found to have profound impacts on diverse cellular processes. Although high-throughput sequencing studies have shown the differential lncRNA expression profiles between hepatocellular carcinoma (HCC) and nontumor livers, the functional impacts of lncRNAs on HCC development await further investigation. Herein, we sought to address the functional roles of lncRNAs in HCC pathogenesis by in vivo functional screening. METHODS: We performed genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/dead CRISPR-associated protein 9 (dCas9) lncRNA activation screening in HCC xenografts. We characterized the clinical relevance of positively selected lncRNAs using transcriptomic data sets. We used CRISPR-based gene activation and knockdown approaches to show the functional roles of positively selected lncRNAs including Cancer Susceptibility 11 (CASC11) in HCC. RNA sequencing and chromatin isolation by RNA purification sequencing were used to investigate the molecular mechanisms of CASC11 in HCC progression. RESULTS: The in vivo functional screening identified 1603 positively selected lncRNAs, 538 of which were overexpressed in HCC patients. Systematic transcriptomic data analysis and clinical investigation showed that patients with high expression of these lncRNA candidates correlated with aggressive tumor behaviors. Overexpression of these lncRNAs aggravated HCC cell growth. Detailed characterization of a lncRNA candidate, CASC11, showed its pivotal role in cell proliferation and tumor growth. Mechanistically, chromatin isolation by RNA purification sequencing showed that CASC11 was bound to the CASC11/MYC proto-oncogene shared promoter region on chromosome 8q24. CASC11 modulated the transcriptional activity of MYC in a cis-regulatory manner, which affected the expression of MYC downstream target genes, consequently promoting G1/S progression. CONCLUSIONS: Our study showed the power of in vivo CRISPR screening, which comprehensively investigated the functionality of lncRNAs in HCC progression, providing a rationale for targeting these lncRNAs clinically.

Generating Custom Pooled CRISPR Libraries for Genetic Dissection of Biological Pathways.

Genetic screens are a classic approach to dissecting biological pathways including membrane trafficking. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 have enabled the utility of this approach in diploid models, including cultured mammalian cells. Here, we present detailed protocols for generating custom CRISPR libraries. These methods are useful for generating genome-wide libraries for new model organisms that lack an existing genome-wide library, and for generating smaller focused libraries.

Cross talk between acetylation and methylation regulators reveals histone modifier expression patterns posing prognostic and therapeutic implications on patients with colon cancer.

BACKGROUND: Alterations in histone modifications have been reported to be related to tumorigenicity and tumor progression. However, whether histone modification can aid the classification of patients or influence clinical behavior in patients with colon cancer remains unclear. Therefore, this study aimed to evaluate histone modifier expression patterns using the unsupervised clustering of the transcriptomic expressions of 88 histone acetylation and methylation regulators. RESULTS: In this study, by consensus clustering analysis based on the transcriptome data of 88 histone modification regulators, we identified four distinct expression patterns of histone modifiers associated with different prognoses, intrinsic fluorouracil sensitivities, biological pathways, and tumor microenvironment characteristics among 1372 colon cancer samples. In these four clusters, the HMC4 cluster represented a stroma activation phenotype characterized by both the worst prognosis and lowest response rates to fluorouracil treatment. Then, we established a scoring scheme comprising 155 genes designated as "HM_score" by using the Boruta algorithm to distinguish colon cancer patients within the HMC4 cluster. Patients with a high HM_score were considered to have high stromal pathway activation, high stromal fraction, and an unfavorable prognosis. Further analyses indicated that a high HM_score also correlated with reduced therapeutic benefits from fluorouracil chemotherapy. Moreover, through CRISPR library screening, ZEB2 was found to be a critical driver gene that mediates fluorouracil resistance, which is associated with histone modifier expression patterns. CONCLUSIONS: This study highlights that characterizing histone modifier expression patterns may help better understand the epigenetic mechanisms underlying tumor heterogeneity in patients with colon cancer and provide more personalized therapeutic strategies.

Evaluation of stromal cell infiltration in the tumor microenvironment enable prediction of treatment sensitivity and prognosis in colon cancer.

Current clinical factors for screening candidates that might benefit from adjuvant chemotherapy in colon cancer are inadequate. Tumor microenvironment, especially the stromal components, has the potential to determine treatment response. However, clinical translation of the tumor-associated stromal characterization into a practical biomarker for helping treatment decision has not been established. Using machine learning, we established a novel 31-gene signature, called stromal cell infiltration intensity score (SIIS), to distinguish patients characterized by the enrichment of abundant stromal cells in five colon cancer datasets from GEO (N = 990). Patients with high-SIIS were at higher risk for recurrence and mortality, and could not benefit from adjuvant chemotherapy due to their intrinsic drug resistance; however, the opposite was reported for patients with low-SIIS. The role of SIIS in detection of patients with high stromal cell infiltration and reduced drug efficiency was consistently validated in the TCGA-COAD cohort (N = 382), Sun Yat-sen University Cancer Center cohort (N = 30), and could also be observed in TCGA pan-cancer settings (N = 4898) and four independent immunotherapy cohorts (N = 467). Based on multi-omics data analysis and the CRISPR library screen, we reported that lack of gene mutation, hypomethylation in ADCY4 promoter region, activation of WNT-PCP pathway and SIAH2-GPX3 axis were potential mechanisms responsible for the chemoresistance of patients within high-SIIS group. Our findings demonstrated that SIIS provide an important reference for those making treatment decisions for such special patients.

CRISPR screens uncover protective effect of PSTK as a regulator of chemotherapy-induced ferroptosis in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is among the most common forms of cancer and is associated with poor patient outcomes. The emergence of therapeutic resistance has hampered the efficacy of targeted treatments employed to treat HCC patients to date. In this study, we conducted a series of CRISPR/Cas9 screens to identify genes associated with synthetic lethality capable of improving HCC patient clinical responses. METHODS: CRISPR-based loss-of-function genetic screens were used to target 18,053 protein-coding genes in HCC cells to identify chemotherapy-related synthetic lethal genes in these cells. Synergistic effects were analyzed through in vitro and in vivo analyses, while related mechanisms were explored through RNA-seq and metabolomics analyses. Potential inhibitors of identified genetic targets were selected through high-throughput virtual screening. RESULTS: The inhibition of phosphoseryl-tRNA kinase (PSTK) was found to increase HCC cell sensitivity to chemotherapeutic treatment. PSTK was associated with the suppression of chemotherapy-induced ferroptosis in HCC cells, and the depletion of PSTK resulted in the inactivation of glutathione peroxidative 4 (GPX4) and the disruption of glutathione (GSH) metabolism owing to the inhibition of selenocysteine and cysteine synthesis, thus enhancing the induction of ferroptosis upon targeted chemotherapeutic treatment. Punicalin, an agent used to treat hepatitis B virus (HBV), was identified as a possible PSTK inhibitor that exhibited synergistic efficacy when applied together with Sorafenib to treat HCC in vitro and in vivo. CONCLUSIONS: These results highlight a key role for PSTK as a mediator of resistance to targeted therapeutic treatment in HCC cells that functions by suppressing ferroptotic induction. PSTK inhibitors may thus represent ideal candidates for overcoming drug resistance in HCC.

A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated gene editing is revolutionizing plant research and crop breeding. Here, we present an effective and streamlined pipeline for arrayed CRISPR library construction and demonstrate it is suitable for small- to large-scale genome editing in plants. This pipeline introduces artificial PCR fragment-length markers for distinguishing guide RNAs (gRNAs) (FLASH), and a group of 12 constructs harboring different FLASH tags are co-transformed into plants each time. The identities of gRNAs in Agrobacterium mixtures and transgenic plants can therefore be read out by detecting the FLASH tags, a process that requires only conventional PCR and gel electrophoresis rather than sequencing. We generated an arrayed CRISPR library targeting all 1,072 members of the receptor-like kinase (RLK) family in rice. One-shot transformation generated a mutant population that covers gRNAs targeting 955 RLKs, and 74.3% (710/955) of the target genes had three or more independent T0 lines. Our results indicate that the FLASH tags act as bona fide surrogates for the gRNAs and are tightly (92.1%) associated with frameshift mutations in the target genes. In addition, the FLASH pipeline allows for rapid identification of unintended editing events without corresponding T-DNA integrations and generates high-order mutants of closely related RLK genes. Furthermore, we showed that the RLK mutant library enables rapid discovery of defense-related RLK genes. This study introduces an effective pipeline for arrayed CRISPR library construction and provides genome-wide rice RLK mutant resources for functional genomics.

Establishment of a pig CRISPR/Cas9 knockout library for functional gene screening in pig cells.

BACKGROUND: As an important farm animal, pig functional genomic study can help understand the molecular mechanism related to the key economic traits of pig, such as growth, reproduction, or disease. The genome-scale library based on clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system facilitates discovery of key genes involved in a specific function or phenotype, allowing for an effective "phenotype-to-genotype" strategy for functional genomic study. METHODS AND RESULTS: We designed and constructed a pig genome-scale CRISPR/Cas9 knockout library targeting 16,888 genes with 970,001 unique sgRNAs. The library is a single-vector system including both Cas9 and sgRNA, and packaged into lentivirus for an easy cell delivery for screening. To establish a screening method in pig cells, we used diphtheria toxin (DT)-induced cell death as a model to screen the host genes critical for DT toxicity in pig PK-15 cells. After lentiviral transduction and two sequential screening with DT treatment, the highest-ranking candidates we identified were previously validated genes, HBEGF, DPH1, DPH2, DPH3, DPH5, DNAJC24, and ZBTB17, which are DT receptor and the key factors involved in biosynthesis of diphthamide, the target of DT action. The function and gene essentiality of candidates were further confirmed by gene knockout and DT toxicity assay in PK-15 cells. CONCLUSIONS: Our CRISPR knockout library targeting pig whole genome establishes a promising platform for pig functional genomic analysis.

The Disulfiram/Copper Complex Induces Autophagic Cell Death in Colorectal Cancer by Targeting ULK1.

Colorectal cancer (CRC) is highly prevalent worldwide, but there has been limited development of efficient and affordable treatment. Induced autophagy has recently been recognized as a novel therapeutic strategy in cancer treatment, and disulfiram (DSF), a well-known antialcohol drug, is also found to inhibit tumor growth in various malignancies. Recently, DSF has been reported to induce excessive autophagy in oral squamous cells; however, little is known about whether it can induce autophagy and suppress proliferation in CRC. In this study, we investigate the effect of DSF with copper (DSF/Cu) on CRC both in vitro and in vivo and find that the combination significantly inhibits CRC cell viability and mainly induces autophagy instead of apoptosis. Furthermore, we use whole genome CRISPR library screening and identify a new mechanism by which DSF triggers autophagy by ULK1. Overall, these findings provide a potential CRC treatment.

WWOX activation by toosendanin suppresses hepatocellular carcinoma metastasis through JAK2/Stat3 and Wnt/ß-catenin signaling.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Loss of WW-domain containing oxidoreductase (WWOX) has been proven to be associated with malignant metastasis in patients with HCC. In this study, by using a non-biased CRISPR knockout genetic screen targeting 19,050 human genes, we found that toosendanin (TSN) is a novel druggable WWOX candidate agonist for metastatic HCC patients. We also found that TSN exhibited significant anti-proliferative and anti-metastatic effects on HCC cells in a WWOX-dependent manner. Overexpression and knockdown of WWOX in vitro and in vivo confirmed that the suppression of HCC by TSN involved WWOX. TSN regulated Stat3, DVL2, and GSK3ß by transforming their interactions with WWOX as demonstrated by a Co-IP assay. TSN accelerated the degradation of ß-catenin by promoting the function of APC, AXIN1, CK1, and GSK3ß complex. Nuclear translocation of p-Stat3 Y705 and ß-catenin was impeded by the TSN-induced blockade of JAK2/Stat3 and Wnt/ß-catenin signaling, accompanied by the inhibition of MMPs and C-MYC.

Genome-wide CRISPR-Cas9 knockout library screening identified PTPMT1 in cardiolipin synthesis is crucial to survival in hypoxia in liver cancer.

Hypoxia, low oxygen (O2), is a key feature of all solid cancers, including hepatocellular carcinoma (HCC). Genome-wide CRISPR-Cas9 knockout library screening is used to identify reliable therapeutic targets responsible for hypoxic survival in HCC. We find that protein-tyrosine phosphatase mitochondrial 1 (PTPMT1), an important enzyme for cardiolipin (CL) synthesis, is the most significant gene and ranks just after hypoxia-inducible factor (HIF)-1α and HIF-1ß as crucial to hypoxic survival. CL constitutes the mitochondrial membrane and ensures the proper assembly of electron transport chain (ETC) complexes for efficient electron transfer in respiration. ETC becomes highly unstable during hypoxia. Knockout of PTPMT1 stops the maturation of CL and impairs the assembly of ETC complexes, leading to further electron leakage and ROS accumulation at ETC in hypoxia. Excitingly, HCC cells, especially under hypoxic conditions, show great sensitivity toward PTPMT1 inhibitor alexidine dihydrochloride (AD). This study unravels the protective roles of PTPMT1 in hypoxic survival and cancer development.

Foxtail Millet: A New Model for C4 Plants.

Arabidopsis and rice are major models for C3 plants, but we still lack a model for C4 plants. Recently, Yang and coworkers developed foxtail millet as a C4 plant model; with the rapid development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas technology, this will open a new era for plant functional studies and crop improvement.

Revealing Temozolomide Resistance Mechanisms via Genome-Wide CRISPR Libraries.

Glioblastoma is a severe type of brain tumor with a poor prognosis and few therapy options. Temozolomide (TMZ) is one of these options, however, with limited success, and failure is mainly due to tumor resistance. In this work, genome-wide CRISPR-Cas9 lentiviral screen libraries for gene knockout or activation were transduced in the human glioblastoma cell line, aiming to identify genes that modulate TMZ resistance. The sgRNAs enriched in both libraries in surviving cells after TMZ treatment were identified by next-generation sequencing (NGS). Pathway analyses of gene candidates on knockout screening revealed several enriched pathways, including the mismatch repair and the Sonic Hedgehog pathways. Silencing three genes ranked on the top 10 list (MSH2, PTCH2, and CLCA2) confirm cell protection from TMZ-induced death. In addition, a CRISPR activation library revealed that NRF2 and Wnt pathways are involved in TMZ resistance. Consistently, overexpression of FZD6, CTNNB1, or NRF2 genes significantly increased cell survival upon TMZ treatment. Moreover, NRF2 and related genes detected in this screen presented a robust negative correlation with glioblastoma patient survival rates. Finally, several gene candidates from knockout or activation screening are targetable by inhibitors or small molecules, and some of them have already been used in the clinic.

Identification of BLNK and BTK as mediators of rituximab-induced programmed cell death by CRISPR screens in GCB-subtype diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is characterized by extensive genetic heterogeneity, and this results in unpredictable responses to the current treatment, R-CHOP, which consists of a cancer drug combination supplemented with the humanized CD20-targeting monoclonal antibody rituximab. Despite improvements in the patient response rate through rituximab addition to the treatment plan, up to 40% of DLBCL patients end in a relapsed or refractory state due to inherent or acquired resistance to the regimen. Here, we employ a lentiviral genome-wide clustered regularly interspaced short palindromic repeats library screening approach to identify genes involved in facilitating the rituximab response in cancerous B cells. Along with the CD20-encoding MS4A1 gene, we identify genes related to B-cell receptor (BCR) signaling as mediators of the intracellular signaling response to rituximab. More specifically, the B-cell linker protein (BLNK) and Bruton's tyrosine kinase (BTK) genes stand out as pivotal genes in facilitating direct rituximab-induced apoptosis through mechanisms that occur alongside complement-dependent cytotoxicity (CDC). Our findings demonstrate that rituximab triggers BCR signaling in a BLNK- and BTK-dependent manner and support the existing notion that intertwined CD20 and BCR signaling pathways in germinal center B-cell-like-subtype DLBCL lead to programmed cell death.

CRISPR screening identifies M1AP as a new MYC regulator with a promoter-reporter system.

BACKGROUND: MYC is one of the proto-oncogenes contributing to tumorigenesis in many human cancers. Although the mechanism of MYC regulation is still not fully understood, learning about the comprehensive mechanism controlling the transcriptional activity of MYC will lead to therapeutic targets. The CRISPR/Cas9 library system is a simple and powerful screening technique. This study aims to identify new transcriptional upstream activators of MYC using the CRISPR activation library with new promoter-reporter systems. METHODS AND RESULTS: The MYC promoter-reporter system was developed with a photoconvertible fluorescent protein, Dendra2, and named "pMYC-promoter-Dendra2." This MYC promoter-reporter system was designed to harbor a proximal MYC promoter at (3.1 kb). Both the CRISPR activation library and pMYC-promoter-Dendra2 were induced to HEK 293T cells, and Dendra2-positive cells, that are supposed that MYC should be upregulated, were collected individually by a cell sorter. Among the 169 cells collected, 12 clones were successfully established. Then, pMYC-promoter-Dendra2 was transfected again into these 12 clones, and two of 12 clones showed Dendra2 positivity. In this procedure, the cells with non-specific autofluorescence were correctly distinguished by utilizing the photoswitchable character of Dendra2. Using extracted genomic DNA of these two Dendra2 positive clones, polymerase chain reaction (PCR) was performed to amplify the guide RNA (gRNA) containing region, which was introduced by the CRISPR activation library. Eventually, PLEKHO2, MICU, MBTPS1, and M1AP were identified, and these gRNAs were transfected individually into HEK 293T cells again using the CRISPR activation system. Only M1AP gRNA transfected cells showed Dendra2-positive fluorescence. Then, the overexpression vector for M1AP with a doxycycline-inducible vector confirmed that M1AP induced high MYC expression by real-time quantitative PCR and western blot. Furthermore, the dual-luciferase assay showed a significant increase of promoter activity, and MYC mRNA was higher in M1AP- overexpressing cells. M1AP is highly expressed in several cancers, though, a positive correlation between M1AP and MYC was observed only in human acute myeloid leukemia. CONCLUSION: The present study confirmed that the experimental method using the CRISPR library technology functions effectively for the identification of molecules that activate endogenous MYC. This method will help elucidate the regulatory mechanism of MYC expression, as well as supporting further drug research against malignant tumors.