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Journal articles on the topic 'CRISPRko Screening'

Author: Grafiati

Published: 1 April 2023

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1

Evers, Bastiaan, Katarzyna Jastrzebski, Jeroen P. M. Heijmans, Wipawadee Grernrum, Roderick L. Beijersbergen, and Rene Bernards. "CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes." Nature Biotechnology 34, no. 6 (April 25, 2016): 631–33. http://dx.doi.org/10.1038/nbt.3536.

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Watters, Kyle E., Christof Fellmann, Hua B. Bai, Shawn M. Ren, and Jennifer A. Doudna. "Systematic discovery of natural CRISPR-Cas12a inhibitors." Science 362, no. 6411 (September 6, 2018): 236–39. http://dx.doi.org/10.1126/science.aau5138.

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Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility for synthetic genome engineering, agricultural genomics, and biomedical applications. Although bacteria harboring CRISPR-Cas9 or CRISPR-Cas3 adaptive immune systems sometimes acquire mobile genetic elements encoding anti-CRISPR proteins that inhibit Cas9, Cas3, or the DNA-binding Cascade complex, no such inhibitors have been found for CRISPR-Cas12a. Here we use a comprehensive bioinformatic and experimental screening approach to identify three different inhibitors that block or diminish CRISPR-Cas12a–mediated genome editing in human cells. We also find a widespread connection between CRISPR self-targeting and inhibitor prevalence in prokaryotic genomes, suggesting a straightforward path to the discovery of many more anti-CRISPRs from the microbial world.

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Selle, Kurt, Todd R. Klaenhammer, and Rodolphe Barrangou. "CRISPR-based screening of genomic island excision events in bacteria." Proceedings of the National Academy of Sciences 112, no. 26 (June 15, 2015): 8076–81. http://dx.doi.org/10.1073/pnas.1508525112.

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Genomic analysis ofStreptococcus thermophilusrevealed that mobile genetic elements (MGEs) likely contributed to gene acquisition and loss during evolutionary adaptation to milk. Clustered regularly interspaced short palindromic repeats–CRISPR-associated genes (CRISPR-Cas), the adaptive immune system in bacteria, limits genetic diversity by targeting MGEs including bacteriophages, transposons, and plasmids. CRISPR-Cas systems are widespread in streptococci, suggesting that the interplay between CRISPR-Cas systems and MGEs is one of the driving forces governing genome homeostasis in this genus. To investigate the genetic outcomes resulting from CRISPR-Cas targeting of integrated MGEs,in silicoprediction revealed four genomic islands without essential genes in lengths from 8 to 102 kbp, totaling 7% of the genome. In this study, the endogenous CRISPR3 type II system was programmed to target the four islands independently through plasmid-based expression of engineered CRISPR arrays. TargetinglacZwithin the largest 102-kbp genomic island was lethal to wild-type cells and resulted in a reduction of up to 2.5-log in the surviving population. Genotyping of Lac−survivors revealed variable deletion events between the flanking insertion-sequence elements, all resulting in elimination of the Lac-encoding island. Chimeric insertion sequence footprints were observed at the deletion junctions after targeting all of the four genomic islands, suggesting a common mechanism of deletion via recombination between flanking insertion sequences. These results established that self-targeting CRISPR-Cas systems may direct significant evolution of bacterial genomes on a population level, influencing genome homeostasis and remodeling.

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Kampmann, Martin, Max A. Horlbeck, Yuwen Chen, Jordan C. Tsai, Michael C. Bassik, Luke A. Gilbert, Jacqueline E. Villalta, et al. "Next-generation libraries for robust RNA interference-based genome-wide screens." Proceedings of the National Academy of Sciences 112, no. 26 (June 15, 2015): E3384—E3391. http://dx.doi.org/10.1073/pnas.1508821112.

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Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.

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Göttl, Vanessa L., Ina Schmitt, Kristina Braun, Petra Peters-Wendisch, Volker F. Wendisch, and Nadja A. Henke. "CRISPRi-Library-Guided Target Identification for Engineering Carotenoid Production by Corynebacterium glutamicum." Microorganisms 9, no. 4 (March 24, 2021): 670. http://dx.doi.org/10.3390/microorganisms9040670.

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Corynebacterium glutamicum is a prominent production host for various value-added compounds in white biotechnology. Gene repression by dCas9/clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) allows for the identification of target genes for metabolic engineering. In this study, a CRISPRi-based library for the repression of 74 genes of C. glutamicum was constructed. The chosen genes included genes encoding enzymes of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, regulatory genes, as well as genes of the methylerythritol phosphate and carotenoid biosynthesis pathways. As expected, CRISPRi-mediated repression of the carotenogenesis repressor gene crtR resulted in increased pigmentation and cellular content of the native carotenoid pigment decaprenoxanthin. CRISPRi screening identified 14 genes that affected decaprenoxanthin biosynthesis when repressed. Carotenoid biosynthesis was significantly decreased upon CRISPRi-mediated repression of 11 of these genes, while repression of 3 genes was beneficial for decaprenoxanthin production. Largely, but not in all cases, deletion of selected genes identified in the CRISPRi screen confirmed the pigmentation phenotypes obtained by CRISPRi. Notably, deletion of pgi as well as of gapA improved decaprenoxanthin levels 43-fold and 9-fold, respectively. The scope of the designed library to identify metabolic engineering targets, transfer of gene repression to stable gene deletion, and limitations of the approach were discussed.

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6

GÜLER KARA, Hale, Buket KOSOVA, Eda DOĞAN, Vildan BOZOK ÇETİNTAŞ, and Şerif ŞENTÜRK. "CRISPR-Cas Functional Genetic Screening: Traditional Review." Turkiye Klinikleri Journal of Medical Sciences 42, no. 4 (2022): 311–22. http://dx.doi.org/10.5336/medsci.2022-88507.

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7

Lanning, Bryan R., and Christopher R. Vakoc. "Single-minded CRISPR screening." Nature Biotechnology 35, no. 4 (April 2017): 339–40. http://dx.doi.org/10.1038/nbt.3849.

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8

Haswell, Jeffrey R., Kaia Mattioli, Chiara Gerhardinger, Philipp G. Maass, Daniel J. Foster, Paola Peinado, Xiaofeng Wang, Pedro P. Medina, John L. Rinn, and Frank J. Slack. "Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency." PLOS ONE 16, no. 11 (November 3, 2021): e0252848. http://dx.doi.org/10.1371/journal.pone.0252848.

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Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.

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9

Ancos-Pintado, Raquel, Irene Bragado-García, María Luz Morales, Roberto García-Vicente, Andrés Arroyo-Barea, Alba Rodríguez-García, Joaquín Martínez-López, María Linares, and María Hernández-Sánchez. "High-Throughput CRISPR Screening in Hematological Neoplasms." Cancers 14, no. 15 (July 25, 2022): 3612. http://dx.doi.org/10.3390/cancers14153612.

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CRISPR is becoming an indispensable tool in biological research, revolutionizing diverse fields of medical research and biotechnology. In the last few years, several CRISPR-based genome-targeting tools have been translated for the study of hematological neoplasms. However, there is a lack of reviews focused on the wide uses of this technology in hematology. Therefore, in this review, we summarize the main CRISPR-based approaches of high throughput screenings applied to this field. Here we explain several libraries and algorithms for analysis of CRISPR screens used in hematology, accompanied by the most relevant databases. Moreover, we focus on (1) the identification of novel modulator genes of drug resistance and efficacy, which could anticipate relapses in patients and (2) new therapeutic targets and synthetic lethal interactions. We also discuss the approaches to uncover novel biomarkers of malignant transformations and immune evasion mechanisms. We explain the current literature in the most common lymphoid and myeloid neoplasms using this tool. Then, we conclude with future directions, highlighting the importance of further gene candidate validation and the integration and harmonization of the data from CRISPR screening approaches.

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Serebrenik, Yevgeniy V., and Ophir Shalem. "CRISPR mutagenesis screening of mice." Nature Cell Biology 20, no. 11 (October 8, 2018): 1235–37. http://dx.doi.org/10.1038/s41556-018-0224-y.

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11

Lau, Esther. "CRISPR screening from both ways." Nature Reviews Genetics 15, no. 12 (October 21, 2014): 778–79. http://dx.doi.org/10.1038/nrg3850.

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12

Eisenstein, Michael. "CRISPR Screening Explores New Dimensions." Genetic Engineering & Biotechnology News 40, no. 7 (July 1, 2020): 26–28. http://dx.doi.org/10.1089/gen.40.07.07.

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13

Zlotorynski, Eytan. "CRISPR–Cas screening for enhancers." Nature Reviews Molecular Cell Biology 17, no. 3 (February 23, 2016): 135. http://dx.doi.org/10.1038/nrm.2016.22.

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14

Watters, Kyle E., Haridha Shivram, Christof Fellmann, Rachel J. Lew, Blake McMahon, and Jennifer A. Doudna. "Potent CRISPR-Cas9 inhibitors fromStaphylococcusgenomes." Proceedings of the National Academy of Sciences 117, no. 12 (March 10, 2020): 6531–39. http://dx.doi.org/10.1073/pnas.1917668117.

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Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibitingStaphylococcus aureusCas9 (SauCas9), an alternative to the most commonly used genome editing proteinStreptococcus pyogenesCas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13–AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13–AcrIIA15 as unique bifunctional inhibitors of SauCas9.

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Camsund, Daniel, Michael J. Lawson, Jimmy Larsson, Daniel Jones, Spartak Zikrin, David Fange, and Johan Elf. "Time-resolved imaging-based CRISPRi screening." Nature Methods 17, no. 1 (November 18, 2019): 86–92. http://dx.doi.org/10.1038/s41592-019-0629-y.

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le Sage, Carlos, Steffen Lawo, and Benedict C. S. Cross. "CRISPR: A Screener’s Guide." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 3 (October 29, 2019): 233–40. http://dx.doi.org/10.1177/2472555219883621.

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The discovery of CRISPR-Cas9 systems has fueled a rapid expansion of gene editing adoption and has impacted pharmaceutical and biotechnology research substantially. Here, gene editing is used at an industrial scale to identify and validate new biological targets for precision medicines, with functional genomic screening having an increasingly important role. Functional genomic strategies provide a crucial link between observed biological phenomena and the genes that influence and drive those phenomena. Although such studies are not new, the use of CRISPR-Cas9 systems in this arena is providing more robust datasets for target identification and validation. CRISPR-based screening approaches are also useful later in the drug development pipeline for understanding drug resistance and sensitivity ahead of entering clinical trials. This review examines the developing landscape for CRISPR screening technologies within the pharmaceutical industry and explores the next steps for this constantly evolving screening platform.

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Степаненко, Liliya Stepanenko, Парамонов, Aleksey Paramonov, Колбасеева, Olga Kolbaseeva, Воскресенская, et al. "BIoInfoRmatIonal analySIS of YersiniapseudotuberculosisIP32953 CRISPR/CaSSyStem." Бюллетень Восточно-Сибирского научного центра Сибирского отделения Российской академии медицинских наук 1, no. 5 (December 6, 2016): 64–67. http://dx.doi.org/10.12737/23384.

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The results of this study include Yersinia pseudotuberculosis CRISPR/Cas system structure analysis. CRISPR/Cas system is a specific adaptive protection against heterogeneous genetic elements. The object of research was the complete genome of Y. pseudotuberculosis IP32953 (NC_006155). CRISPR/Cas system screening was performed by program modelling methods MacSyFinder ver. 1.0.2. CRISPR loci screening and analyzing were carried out by program package: CRISPR Recognition tool (CRT), CRISPI: a CRISPR Interactive database, CRISPRFinder, and PilerCR. Spacer sequences were used in order to find protospacers in ACLAME, GenBank-Phage and RefSeq-Plasmid databases by BLASTn search algorithm. Protospacer sequences could be found in genomes of phages, plasmids and bacteria. In last case complete genomes of bacteria were analyzed by online-tool PHAST: PHAge Search Tool. Y. pseudotuberculosis IP329353 has CRISPR/Cas system that consists of one sequence of cas-genes and three loci. These loci are far away from each other. Locus YP1 is situated in close proximity to cas-genes. Protospacers were found in genomes of Y. pseudotuberculosis PB1/+, Y. intermedia Y228, Y. similis str. 228, Salmonella phage, Enterobacteria phage, Y. pseudotuberculosis IP32953 plasmid pYV and plasmid of Y. pseudotuberculosis IP31758. Thus, the combination of four program methods allows finding CRISPR/Cas system more precisely. Spacer sequences could be used for protospacer screening.

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Yin, Zixi, and Lingyi Chen. "Simple Meets Single: The Application of CRISPR/Cas9 in Haploid Embryonic Stem Cells." Stem Cells International 2017 (2017): 1–6. http://dx.doi.org/10.1155/2017/2601746.

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The CRISPR/Cas9 system provides a powerful method for the genetic manipulation of the mammalian genome, allowing knockout of individual genes as well as the generation of genome-wide knockout cell libraries for genetic screening. However, the diploid status of most mammalian cells restricts the application of CRISPR/Cas9 in genetic screening. Mammalian haploid embryonic stem cells (haESCs) have only one set of chromosomes per cell, avoiding the issue of heterozygous recessive mutations in diploid cells. Thus, the combination of haESCs and CRISPR/Cas9 facilitates the generation of genome-wide knockout cell libraries for genetic screening. Here, we review recent progress in CRISPR/Cas9 and haPSCs and discuss their applications in genetic screening.

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Choi, Ahyoung, Insu Jang, Heewon Han, Min-Seo Kim, Jinhyuk Choi, Jieun Lee, Sung-Yup Cho, et al. "iCSDB: an integrated database of CRISPR screens." Nucleic Acids Research 49, no. D1 (November 2, 2020): D956—D961. http://dx.doi.org/10.1093/nar/gkaa989.

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Abstract High-throughput screening based on CRISPR-Cas9 libraries has become an attractive and powerful technique to identify target genes for functional studies. However, accessibility of public data is limited due to the lack of user-friendly utilities and up-to-date resources covering experiments from third parties. Here, we describe iCSDB, an integrated database of CRISPR screening experiments using human cell lines. We compiled two major sources of CRISPR-Cas9 screening: the DepMap portal and BioGRID ORCS. DepMap portal itself is an integrated database that includes three large-scale projects of CRISPR screening. We additionally aggregated CRISPR screens from BioGRID ORCS that is a collection of screening results from PubMed articles. Currently, iCSDB contains 1375 genome-wide screens across 976 human cell lines, covering 28 tissues and 70 cancer types. Importantly, the batch effects from different CRISPR libraries were removed and the screening scores were converted into a single metric to estimate the knockout efficiency. Clinical and molecular information were also integrated to help users to select cell lines of interest readily. Furthermore, we have implemented various interactive tools and viewers to facilitate users to choose, examine and compare the screen results both at the gene and guide RNA levels. iCSDB is available at https://www.kobic.re.kr/icsdb/.

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Liu, S. John, Max A. Horlbeck, Seung Woo Cho, Harjus S. Birk, Martina Malatesta, Daniel He, Frank J. Attenello, et al. "CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells." Science 355, no. 6320 (December 15, 2016): eaah7111. http://dx.doi.org/10.1126/science.aah7111.

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The human genome produces thousands of long noncoding RNAs (lncRNAs)—transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type–specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs.

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Raffeiner, Philipp, Jonathan R. Hart, Daniel García-Caballero, Liron Bar-Peled, Marc S. Weinberg, and Peter K. Vogt. "An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation." Proceedings of the National Academy of Sciences 117, no. 12 (March 10, 2020): 6571–79. http://dx.doi.org/10.1073/pnas.1921786117.

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MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.

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Thege, Fredrik Ivar, Dhwani N. Rupani, Bhargavi B. Barathi, Anirban Maitra, Andrew D. Rhim, and Sonja M. Wörmann. "Abstract 918: Development of a platform for programmable in vivo oncogene activation and screening using CRISPRa technology." Cancer Research 82, no. 12_Supplement (June 15, 2022): 918. http://dx.doi.org/10.1158/1538-7445.am2022-918.

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Abstract Conventional genetically engineered mouse models (GEMMs) are time consuming, laborious and offer limited spatio-temporal control. We have developed a streamlined platform for in vivo gene activation using CRISPR activation (CRISPRa) technology. Our model system allows for flexible, sustained and timed activation of one or more target genes, in vitro or in vivo, using single or pooled lentiviral guides. Using Myc and Yap1 as model oncogenes, we implemented this platform to study the effect of oncogene activation on the tumorigenic potential of primary pancreatic organoids, as well as in an autochthonous model of lung adenocarcinoma. We found that Myc-activation in pancreatic organoids increased their tumorigenic potential and resulted in significantly shorter survival relative to controls when transplanted orthotopically. In vivo Myc activation in the lung accelerated tumor progression and resulted in significantly shorter overall survival relative to non-targeted tumor controls. Furthermore, we found that Myc-activation drives the acquisition of an immune suppressive “cold” tumor microenvironment. Through cross-species validation of our results, we were able to link MYC to a previously described, immunosuppressive transcriptomic subtype in patient tumors, thus identifying a patient cohort that may benefit from combined MYC/immune-targeted therapies. Our work demonstrates how CRISPRa can be used for rapid functional validation of putative oncogenes and may allow for the identification and evaluation of potential metastatic and oncogenic drivers through competitive screening. Citation Format: Fredrik Ivar Thege, Dhwani N. Rupani, Bhargavi B. Barathi, Anirban Maitra, Andrew D. Rhim, Sonja M. Wörmann. Development of a platform for programmable in vivo oncogene activation and screening using CRISPRa technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 918.

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Winkless, Laurie. "High-throughput screening platform for CRISPR." Materials Today 19, no. 3 (April 2016): 132. http://dx.doi.org/10.1016/j.mattod.2016.02.017.

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Salzman, Sony. "How CRISPR Is Revolutionizing Screening Technology." Genetic Engineering & Biotechnology News 39, no. 4 (April 2019): S16—S18. http://dx.doi.org/10.1089/gen.39.04.23.

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Salzman, Sony. "How CRISPR Is Revolutionizing Screening Technology." Genetic Engineering & Biotechnology News 39, S2 (April 2019): S16—S18. http://dx.doi.org/10.1089/gen.39.s2.06.

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Liu, Zhuoxin. "CRISPR/Cas9 high-throughput screening in cancer research." E3S Web of Conferences 185 (2020): 03032. http://dx.doi.org/10.1051/e3sconf/202018503032.

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In recent years, CRISPR/Cas9 technology has developed rapidly. With its accurate, fast, and simple editing functions that can achieve gene activation, interference, knockout, and knock-in, it has become a powerful genetic screening tool that is widely used in various models, including cell lines of mice and zebrafish. The use of CRISPR system to construct a genomic library for high-throughput screening is the main strategy for research of disease, especially tumor target gene research. This article reviews the basic principles and latest developments of CRISPR/Cas9 library screening technology strategies to improve its off-target effect, the basic workflow of library screening, and its application in tumor research.

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Tsung, Kathleen, Jane Han, Kristie Liu, Eddie Loh, and Frank Attenello. "CNSC-31. CRISPR FUNCTIONAL SCREEN IDENTIFIES A NOVEL LONG NONCODING RNA MODULATING GLIOBLASTOMA INVASION." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii29. http://dx.doi.org/10.1093/neuonc/noac209.112.

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Abstract INTRODUCTION Glioblastoma(GBM) invasion studies traditionally target coding genes. Long noncoding RNAs (lncRNAs), are transcripts without coding potential, with limited data regarding function. We leveraged CRISPR-interference, repressing lncRNA transcription via Cas9-KRAB, to evaluate invasive function of GBM-associated lncRNAs in a CRISPRi functional screen. METHODS 2,307 candidate lncRNAs were assembled from GBM differential gene expression. Guide RNAs targeting candidates were lentivirally incorporated into GBM screening populations. Screen populations were evaluated via Matrigel invasion, comparing sgRNA populations before/after a 24-hour invasion period. ASO treatment evaluated pharmacologic targeting of candidate lncRNAs. Top screening candidates were evaluated in a patient derived glioma stem cell (GSC) line, USC02, as well as U87 and U251 commercial lines. Differential gene expression was assessed in multiple cell lines following lncRNA knockdown via RNA-seq followed by GSEA and pathway analysis was evaluated to assess downstream mechanistic candidates affected by lncRNA KD. RESULTS Forty-eight lncRNAs were significantly associated with GBM invasion, with lncRNA repression associated with decreased invasion of 31-85%(p< 0.01). Individual validation of a candidate lncRNA KD, LH02236, confirmed 85% decrease in tumor cell invasion versus control. Evaluation of gene expression across normal cortex and tumor samples from GSEA/TCGA datasets revealed LH02236 is significantly more expressed in GBM versus low grade glioma, and in low grade glioma versus normal cortex. Expression of LH02236 was significantly associated with patient survival (p< 0.0001). Gene expression analysis of LH02236 knockdown revealed Sox10 to be highly correlated with lncRNA expression across multiple cell lines. CONCLUSION Large scale CRISPRi screening identified LH02236, a previously unannotated lncRNA, as essential to invasion in patient lines. Gene expression is significantly associated with tumor grade and patient survival. Analysis of knockdown across multiple cell lines suggests Sox 10, commonly associated with GSC function, is modulated by this novel lncRNA.

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Chulanov, Vladimir, Anastasiya Kostyusheva, Sergey Brezgin, Natalia Ponomareva, Vladimir Gegechkori, Elena Volchkova, Nikolay Pimenov, and Dmitry Kostyushev. "CRISPR Screening: Molecular Tools for Studying Virus–Host Interactions." Viruses 13, no. 11 (November 11, 2021): 2258. http://dx.doi.org/10.3390/v13112258.

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CRISPR/Cas is a powerful tool for studying the role of genes in viral infections. The invention of CRISPR screening technologies has made it possible to untangle complex interactions between the host and viral agents. Moreover, whole-genome and pathway-specific CRISPR screens have facilitated identification of novel drug candidates for treating viral infections. In this review, we highlight recent developments in the fields of CRISPR/Cas with a focus on the use of CRISPR screens for studying viral infections and identifying new candidate genes to aid development of antivirals.

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Chen, Sitong, Lin Yang, and Wei Li. "CRISPR Screening “Big Data” Informs Novel Therapeutic Solutions." CRISPR Journal 2, no. 3 (June 2019): 152–54. http://dx.doi.org/10.1089/crispr.2019.29062.sch.

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Shaffer, Catherine. "CRISPR's Rapid Rise Shakes Up Genome-Wide Screening." Genetic Engineering & Biotechnology News 41, no. 5 (May 1, 2021): 46–49. http://dx.doi.org/10.1089/gen.41.05.13.

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Cao, Qingyi, Jian Ma, Chen-Hao Chen, Han Xu, Zhi Chen, Wei Li, and X. Shirley Liu. "CRISPR-FOCUS: A web server for designing focused CRISPR screening experiments." PLOS ONE 12, no. 9 (September 5, 2017): e0184281. http://dx.doi.org/10.1371/journal.pone.0184281.

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Zhou, Peng, Yuk Kei Wan, Becky K. C. Chan, Gigi C. G. Choi, and Alan S. L. Wong. "Extensible combinatorial CRISPR screening in mammalian cells." STAR Protocols 2, no. 1 (March 2021): 100255. http://dx.doi.org/10.1016/j.xpro.2020.100255.

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Housden, Benjamin E., and Norbert Perrimon. "Comparing CRISPR and RNAi-based screening technologies." Nature Biotechnology 34, no. 6 (June 2016): 621–23. http://dx.doi.org/10.1038/nbt.3599.

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Wang, William, and Xiangdong Wang. "Single-cell CRISPR screening in drug resistance." Cell Biology and Toxicology 33, no. 3 (May 4, 2017): 207–10. http://dx.doi.org/10.1007/s10565-017-9396-7.

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35

Shen, ZhongFu, and GuangShuo Ou. "CRISPR-Cas9 knockout screening for functional genomics." Science China Life Sciences 57, no. 7 (June 10, 2014): 733–34. http://dx.doi.org/10.1007/s11427-014-4684-4.

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36

Sobh, Amin, and Chris Vulpe. "CRISPR genomic screening informs gene–environment interactions." Current Opinion in Toxicology 18 (December 2019): 46–53. http://dx.doi.org/10.1016/j.cotox.2019.02.009.

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37

LaFlamme, Brooke. "A CRISPR method for genome-wide screening." Nature Genetics 46, no. 2 (January 29, 2014): 99. http://dx.doi.org/10.1038/ng.2887.

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Schmierer, Bernhard, Sandeep K. Botla, Jilin Zhang, Mikko Turunen, Teemu Kivioja, and Jussi Taipale. "CRISPR/Cas9 screening using unique molecular identifiers." Molecular Systems Biology 13, no. 10 (October 2017): 945. http://dx.doi.org/10.15252/msb.20177834.

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39

Yang, Bing, and Katherine McJunkin. "CRISPR screening strategies for microRNA target identification." FEBS Journal 287, no. 14 (February 6, 2020): 2914–22. http://dx.doi.org/10.1111/febs.15218.

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40

Hong, Lemin, Chenlu Zhang, Yijing Jiang, Haiyan Liu, Hongming Huang, and Dan Guo. "Therapeutic status and the prospect of CRISPR/Cas9 gene editing in multiple myeloma." Future Oncology 16, no. 16 (June 2020): 1125–36. http://dx.doi.org/10.2217/fon-2019-0822.

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In recent years, CRISPR/Cas9, a novel gene-editing technology, has shown considerable potential in the design of novel research methods and future options for treating multiple myeloma (MM). The use of CRISPR/Cas9 promises faster and more accurate identification and validation of target genes. In this review, we summarize the current research status of the application of CRISPR technology in MM, especially in detecting the expression of MM gene, exploring the mechanism of drug action, screening for drug-resistant genes, developing immunotherapy and screening for new drug targets. Given the tremendous progress that has been made, we believe that CRISPR/Cas9 possesses great potential in MM-related clinical practice.

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Neff, Ellen. "CRISPRa screening in mice for melanoma’s Achilles’ heel." Lab Animal 50, no. 5 (April 19, 2021): 122. http://dx.doi.org/10.1038/s41684-021-00762-7.

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42

Xu, Chunlong, Xiaolan Qi, Xuguang Du, Huiying Zou, Fei Gao, Tao Feng, Hengxing Lu, et al. "piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice." Proceedings of the National Academy of Sciences 114, no. 4 (January 6, 2017): 722–27. http://dx.doi.org/10.1073/pnas.1615735114.

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CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and unknown tumor suppressor genes (TSGs). Our results demonstrate that PB can be a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries.

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Madsen, Ralitsa R., and Robert K. Semple. "Luminescent peptide tagging enables efficient screening for CRISPR-mediated knock-in in human induced pluripotent stem cells." Wellcome Open Research 4 (February 20, 2019): 37. http://dx.doi.org/10.12688/wellcomeopenres.15119.1.

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Human pluripotent stem cells are increasingly used for CRISPR-mediated gene targeting in efforts to generate models of human diseases. This is a challenging task because of the high sensitivity of these cells to suboptimal conditions, including CRISPR-associated DNA damage and subsequent rounds of single-cell cloning. We sought to develop a sensitive method that enables rapid screening of CRISPR targeted cells, while preserving cell viability and eliminating the need for expensive sequencing of a large number of clones. A protocol was designed in which the luminescent peptide tag, HiBiT, is appended to the extracellular portion of an inert surface membrane protein (CD46), using synthetic CRISPR reagents and a widely distributed human induced pluripotent stem cell (iPSC) line. We find that this approach substantially reduces labour-intensive screening of CRISPR-targeted iPSCs and minimises the number of subcloning steps. Successfully edited iPSCs could be identified within a week of targeting, based only on extracellular luminescence detection in live cells. The total screening time in each round was less than 30 minutes and no sequencing was required. This method can be developed further to serve as a highly sensitive co-selection strategy in CRISPR knock-in experiments, particularly in the context of challenging cell lines.

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Madsen, Ralitsa R., and Robert K. Semple. "Luminescent peptide tagging enables efficient screening for CRISPR-mediated knock-in in human induced pluripotent stem cells." Wellcome Open Research 4 (April 15, 2019): 37. http://dx.doi.org/10.12688/wellcomeopenres.15119.2.

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Abstract:

Human pluripotent stem cells are increasingly used for CRISPR-mediated gene targeting in efforts to generate models of human diseases. This is a challenging task because of the high sensitivity of these cells to suboptimal conditions, including CRISPR-associated DNA damage and subsequent rounds of single-cell cloning. We sought to develop a sensitive method that enables rapid screening of CRISPR targeted cells, while preserving cell viability and eliminating the need for expensive sequencing of a large number of clones. A protocol was designed in which the luminescent peptide tag, HiBiT, is appended to the extracellular portion of an inert surface membrane protein (CD46), using synthetic CRISPR reagents and a widely distributed human induced pluripotent stem cell (iPSC) line. We find that this approach substantially reduces labour-intensive screening of CRISPR-targeted iPSCs and minimises the number of subcloning steps. Successfully edited iPSCs could be identified within a week of targeting, based only on extracellular luminescence detection in live cells. The total screening time in each round was less than 30 minutes and no sequencing was required. This method can be developed further to serve as a highly sensitive co-selection strategy in CRISPR knock-in experiments, particularly in the context of challenging cell lines.

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45

Madsen, Ralitsa R., and Robert K. Semple. "Luminescent peptide tagging enables efficient screening for CRISPR-mediated knock-in in human induced pluripotent stem cells." Wellcome Open Research 4 (July 11, 2019): 37. http://dx.doi.org/10.12688/wellcomeopenres.15119.3.

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Abstract:

Human pluripotent stem cells are increasingly used for CRISPR-mediated gene targeting in efforts to generate models of human diseases. This is a challenging task because of the high sensitivity of these cells to suboptimal conditions, including CRISPR-associated DNA damage and subsequent rounds of single-cell cloning. We sought to develop a sensitive method that enables rapid screening of CRISPR targeted cells, while preserving cell viability and eliminating the need for expensive sequencing of a large number of clones. A protocol was designed in which the luminescent peptide tag, HiBiT, is appended to the extracellular portion of an inert surface membrane protein (CD46), using synthetic CRISPR reagents and a widely distributed human induced pluripotent stem cell (iPSC) line. We find that this approach substantially reduces labour-intensive screening of CRISPR-targeted iPSCs and minimises the number of subcloning steps. Successfully edited iPSCs could be identified within a week of targeting, based only on extracellular luminescence detection in live cells. The total screening time in each round was less than 30 minutes and no sequencing was required. This method can be developed further to serve as a highly sensitive co-selection strategy in CRISPR knock-in experiments, particularly in the context of challenging cell lines.

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Benbarche, Salima, Cécile K. Lopez, Thomas Mercher, and Camille Lobry. "Crispri-Based Screening of Clustered Regulatory Elements Reveals Novel Leukemia Dependencies." Blood 132, Supplement 1 (November 29, 2018): 654. http://dx.doi.org/10.1182/blood-2018-99-111865.

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Abstract In the recent years, massively parallel sequencing approaches allowed the identification of hundreds of mutated genes in Leukemia. Although these data gave unprecedented amount of information about mechanisms of leukemia cell maintenance and/or progression, the functional characterization of genes that are key player in regulating cancer development remain laborious. Analysis at the single gene level often fails to identify gene or pathway collaborations leading to transformation. Studies aimed at depicting new oncogene cooperation would involve the generation of challenging mouse models or the deployment of tedious screening pipelines, which would be inadequate to depict new oncogene circuitry in cancer. Genome wide mapping of epigenetic modifications on histone tails or binding of factors such as MED1 and BRD4 allowed identification of clusters of regulatory elements, also termed as Super Enhancers. Functional annotation of these regions revealed their high relevance during normal hematopoiesis and Leukemogenesis. We hypothesized that these regulatory regions could regulate simultaneously expression of genes cooperating to promote Leukemia development. We thus developed a novel genome-wide CRISPRi-based screening approach to directly target these regulatory regions. CRISPRi technology relies on the use of deactivated Cas9 that can't cut the DNA and that is fused to the repressive KRAB domain (dCas9-KRAB). Therefore, properly targeted dCas9-KRAB by single guide RNAs will recruit chromatin modifying factors and trigger generation of heterochromatin thus inhibiting enhancer function. We performed this screen using acute megakaryoblastic leukemia model driven by the CBFA2T3-GLIS2 fusion, the most frequent fusion oncogene in this disease that we recently identified as being associated with Super Enhancers (Thirant et al, Cancer Cell 2017). To inhibit Super Enhancer activity we integrated ChIP-seq data of H3K27ac and ATAC-seq data to define open chromatin regions located in Super Enhancers. We designed a library of 7995 single guide RNAs targeting 450 Super Enhancer regions found active in CBFA2T3-GLIS2 bearing cell line M07e and primary AMKL patient samples. This screening methodology allowed us to nominate Super Enhancer regions, which are functionally linked to leukemia progression. In particular, we pinpointed a novel Super Enhancer region, induced by CBFA2T3-GLIS2 fusion, regulating the expression of both tyrosine kinases associated receptors KIT and PDGFRA. We were able to show that this Super Enhancer region is normally not active in normal megakaryocytic development and aberrantly induced by CBFA2T3-GLIS2 expression. RNA-sequencing experiments and 4C-seq experiments (chromatin conformation capture) showed that this Super Enhancer is directly regulating KIT and PDGFRA expression. Whereas single inhibition of these genes using shRNA or small molecule inhibitors affects modestly leukemic cell growth, concomitant inhibition of these two receptors synergizes to impair AMKL cell lines and primary patient cells growth and survival. In vivo targeting of this Super Enhancer activity in patient-derived xenograft models using CRISPRi showed significant reduction of tumor burden and increased overall survival. Our results demonstrate that genome-wide screening of regulatory DNA elements can identify co-regulated genes collaborating to promote leukemia progression and could open new avenues for the design of combination therapies. Reference: Thirant C, Ignacimouttou C, Lopez CK, Diop M, Le Mouël L, Thiollier C, Siret A, Dessen P, Aid Z, Rivière J, Rameau P, Lefebvre C, Khaled M, Leverger G, Ballerini P, Petit A, Raslova H, Carmichael CL, Kile BT, Soler E, Crispino JD, Wichmann C, Pflumio F, Schwaller J, Vainchenker W, Lobry C, Droin N, Bernard OA, Malinge S, Mercher T (2017). ETO2-GLIS2 Hijacks Transcriptional Complexes to Drive Cellular Identity and Self-Renewal in Pediatric Acute Megakaryoblastic Leukemia. Cancer Cell. 31(3):452-465. Disclosures No relevant conflicts of interest to declare.

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47

Wong, Alan S. L., Gigi C. G. Choi, Cheryl H. Cui, Gabriela Pregernig, Pamela Milani, Miriam Adam, Samuel D. Perli, et al. "Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM." Proceedings of the National Academy of Sciences 113, no. 9 (February 10, 2016): 2544–49. http://dx.doi.org/10.1073/pnas.1517883113.

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The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas–based knockouts and RNA-interference–based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.

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Veeneman, Brendan, Ying Gao, Joy Grant, David Fruhling, James Ahn, Benedikt Bosbach, Jadwiga Bienkowska, et al. "PINCER: improved CRISPR/Cas9 screening by efficient cleavage at conserved residues." Nucleic Acids Research 48, no. 17 (August 21, 2020): 9462–77. http://dx.doi.org/10.1093/nar/gkaa645.

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Abstract CRISPR/Cas9 functional genomic screens have emerged as essential tools in drug target discovery. However, the sensitivity of available genome-wide CRISPR libraries is impaired by guides which inefficiently abrogate gene function. While Cas9 cleavage efficiency optimization and essential domain targeting have been developed as independent guide design rationales, no library has yet combined these into a single cohesive strategy to knock out gene function. Here, in a massive reanalysis of CRISPR tiling data using the most comprehensive feature database assembled, we determine which features of guides and their targets best predict activity and how to best combine them into a single guide design algorithm. We present the ProteIN ConsERvation (PINCER) genome-wide CRISPR library, which for the first time combines enzymatic efficiency optimization with conserved length protein region targeting, and also incorporates domains, coding sequence position, U6 termination (TTT), restriction sites, polymorphisms and specificity. Finally, we demonstrate superior performance of the PINCER library compared to alternative genome-wide CRISPR libraries in head-to-head validation. PINCER is available for individual gene knockout and genome-wide screening for both the human and mouse genomes.

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Dong, Matthew B., Kaiyuan Tang, Xiaoyu Zhou, Jingjia J. Zhou, and Sidi Chen. "Tumor immunology CRISPR screening: present, past, and future." Trends in Cancer 8, no. 3 (March 2022): 210–25. http://dx.doi.org/10.1016/j.trecan.2021.11.009.

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50

Shah, Arish N., Crystal F. Davey, Alex C. Whitebirch, Adam C. Miller, and Cecilia B. Moens. "Rapid reverse genetic screening using CRISPR in zebrafish." Nature Methods 12, no. 6 (April 13, 2015): 535–40. http://dx.doi.org/10.1038/nmeth.3360.

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