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CRISPR selection

Philippe C Després, Alexandre K Dubé, Lou Nielly-Thibault, Nozomu Yachie, Christian R Landry
CRISPR-Cas9 loss of function (LOF) and base editing screens are powerful tools in genetics and genomics. Yeast is one of the main models in these fields, but has only recently started to adopt this new toolkit for high throughput experiments. We developed a double selection strategy based on co-selection that increases LOF mutation rates using the Target-AID base editor. We constructed the pDYSCKO vector, which is amenable to high throughput double selection experiments, and show that the improvement in Target-AID efficiency generalizes across loci...
August 10, 2018: G3: Genes—Genomes—Genetics
Wookjae Lee, Joon Ho Lee, Soyeong Jun, Ji Hyun Lee, Duhee Bang
Mutations within the KRAS oncogene are associated with the proliferation of various cancers. Therapeutic approaches for treating cancers with such mutations have focused on targeting the downstream protein effectors of KRAS. However, to date, no approved treatment has targeted the mutated KRAS oncogene directly. Presently, we used the selectivity of the CRISPR/Cas9 system to directly target mutated KRAS alleles. We designed single-guide RNAs (sgRNAs) to target two specific single-nucleotide missense mutations on KRAS codon-12 located in the seed region adjacent to a protospacer adjacent motif (PAM)...
August 8, 2018: Scientific Reports
Zachary WareJoncas, Jarryd M Campbell, Gabriel Martínez-Gálvez, William A C Gendron, Michael A Barry, Peter C Harris, Caroline R Sussman, Stephen C Ekker
The expanding field of precision gene editing is empowering researchers to directly modify DNA. Gene editing is made possible using synonymous technologies: a DNA-binding platform to molecularly locate user-selected genomic sequences and an associated biochemical activity that serves as a functional editor. The advent of accessible DNA-targeting molecular systems, such as zinc-finger nucleases, transcription activator-like effectors (TALEs) and CRISPR-Cas9 gene editing systems, has unlocked the ability to target nearly any DNA sequence with nucleotide-level precision...
August 8, 2018: Nature Reviews. Nephrology
Xinyi Li, Yichun Bai, Xinzhen Cheng, Peter Girgis Tawfek Kalds, Bing Sun, Yun Wu, Huijiao Lyu, Kun Xu, Zhiying Zhang
CRISPR/Cas9 has been emerging as a main player in genome editing field since its advent. However, CRISPR/Cas9-induced precise gene editing remains challenging since it requires no scar left after editing. Among the few reports regarding two-step 'pop in & out' technologies for precise gene editing, the combination of CRISPR/Cas9 with Cre/LoxP demonstrates a higher efficiency, but leaves behind a 34 base pair of tag sequence due to its inherent property. Another method utilizes piggyBac transposon for removing the selection cassette, and its disadvantage is the difficulty in controlling its random reintegration after releasing...
August 7, 2018: FEBS Journal
Nicolas Jardin, François Giudicelli, Daniel Ten Martín, Anaïs Vitrac, Stéphanie De Gois, Rachel Allison, Corinne Houart, Evan Reid, Jamilé Hazan, Coralie Fassier
Functional analyses of genes responsible for neurodegenerative disorders have unveiled critical links between neurodegenerative processes and key developmental signalling pathways. Mutations in SPG4 encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes coupling microtubule severing to membrane remodelling. Two main spastin isoforms are synthesised from alternative translational start sites (M1 and M87). However, their specific roles in neuronal development and homeostasis remain largely unknown...
August 6, 2018: Development
Ilya Blokhin, Olga Khorkova, Jane Hsiao, Claes Wahlestedt
The central dogma of molecular biology, which states that the only role of long RNA transcripts is to convey information from gene to protein, was brought into question in recent years due to discovery of the extensive presence and complex roles of long noncoding RNAs (lncRNAs). Furthermore, lncRNAs were found to be involved in pathogenesis of multiple diseases and thus represent a new class of therapeutic targets. Translational efforts in the lncRNA field have been augmented by progress in optimizing the chemistry and delivery platforms of lncRNA-targeting modalities, including oligonucleotide-based drugs and CRISPR-Cas9...
August 6, 2018: Expert Opinion on Drug Discovery
Brandon W Simone, Gabriel Martínez-Gálvez, Zachary WareJoncas, Stephen C Ekker
The rapid growth of the field of gene editing can largely be attributed to the discovery and optimization of designer endonucleases. These include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regular interspersed short palindromic repeat (CRISPR) systems including Cas9, Cas12a, and structure-guided nucleases. Zebrafish (Danio rerio) have proven to be a powerful model system for genome engineering testing and applications due to their external development, high fecundity, and ease of housing...
August 1, 2018: Methods: a Companion to Methods in Enzymology
Xiaohu Su, Shenyuan Wang, Guanghua Su, Zhong Zheng, Jiaqi Zhang, Yunlong Ma, Zongzheng Liu, Huanmin Zhou, Yanru Zhang, Li Zhang
Gene editing tools (Zinc-Finger Nucleases, ZFN; Transcription Activator-Like Effector Nucleases, TALEN; and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)9, CRISPR-Cas9) provide us with a powerful means of performing genetic engineering procedures. A combinational approach that utilizes both somatic cell nuclear transfer (SCNT) and somatic cell gene editing facilitates the generation of genetically engineered animals. However, the associated research has utilized markers and/or selected genes, which constitute a potential threat to biosafety...
July 20, 2018: Theriogenology
Yan-Xiang Cheng, Gan-Tao Chen, Xiao Yang, Yan-Qing Wang, Li Hong
The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer. After designing specific gRNA sequences targeting HPV16 E6, generating hCas9-EGFP and E6-gRNA-RFP plasmids, and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids, we determined the titer of the pseudotype virus using the TCID50 method...
April 2018: Current medical science
Yanglei Yi, Zhibo Li, Chunxu Song, Oscar P Kuipers
Bacillus subtilis HS3 and Bacillus mycoides EC18 are two rhizosphere-associated bacteria with plant growth-promoting activity. The CRISPR-Cas9 system was implemented to study various aspects of plant-microbe interaction mechanisms of these two environmental isolates. The results show that fengycin and surfactin are involved in the antifungal activity of B. subtilis HS3. Moreover, this strain emits several other volatile organic compounds (VOCs) than 2,3-butanediol, contributing to plant growth promotion. CLSM (Confocal laser scanning microscopy) observations of the GFP-labelled strain showed that HS3 selectively colonizes root hairs of grass (Lolium perenne) in a hydroponic system...
July 26, 2018: Environmental Microbiology
Jacob C Garza, Xiaoli Qi, Klaudio Gjeluci, Melanie P Leussis, Himanish Basu, Surya A Reis, Wen Ning Zhao, Nicolas H Piguel, Peter Penzes, Stephen J Haggarty, Gerard J Martens, Geert Poelmans, Tracey L Petryshen
The ankyrin 3 gene (ANK3) is a well-established risk gene for psychiatric illness, but the mechanisms underlying its pathophysiology remain elusive. We examined the molecular effects of disrupting brain-specific Ank3 isoforms in mouse and neuronal model systems. RNA sequencing of hippocampus from Ank3+/- and Ank3+/+ mice identified altered expression of 282 genes that were enriched for microtubule-related functions. Results were supported by increased expression of microtubule end-binding protein 3 (EB3), an indicator of microtubule dynamics, in Ank3+/- mouse hippocampus...
July 25, 2018: Translational Psychiatry
Shigeo S Sugano, Ryuichi Nishihama
Plant transcription factors (TFs) belong to a wide variety of gene families. The systematic and rapid establishment of knockout lines of TF genes is critical for functional genetics. Genome engineering techniques for dissecting out the molecular function of TFs have been dramatically improved by CRISPR/Cas9-based genome editing technology. In the CRISPR/Cas9 system, Cas9 functions as a Cas9-gRNA ribonucleoprotein complex and uses its DNA endonuclease activity to induce the cleavage of the genome, which is targeted by gRNA...
2018: Methods in Molecular Biology
Quinlan L Sievers, Jessica A Gasser, Glenn S Cowley, Eric S Fischer, Benjamin L Ebert
Lenalidomide mediates the ubiquitination and degradation of IKZF1, IKZF3 and CK1α by facilitating their interaction with cereblon (CRBN), the substrate receptor for the CRL4CRBN E3 ubiquitin ligase. Through this mechanism, lenalidomide is a clinically effective treatment for multiple myeloma and myelodysplastic syndrome with deletion of chromosome 5q (del(5q) MDS). To identify the cellular machinery required for lenalidomide-induced CRL4CRBN activity, we performed a positive selection, genome-scale CRISPR-Cas9 screen in a lenalidomide-sensitive myeloma cell line...
July 24, 2018: Blood
Navin Gupta, Koichiro Susa, Yoko Yoda, Joseph V Bonventre, M Todd Valerius, Ryuji Morizane
Human pluripotent stem cells (hPSCs) represent a formidable tool for disease modeling, drug discovery, and regenerative medicine using human cells and tissues in vitro. Evolving techniques of targeted genome editing, specifically the CRISPR/Cas9 system, allow for the generation of cell lines bearing gene-specific knock-outs, knock-in reporters, and precise mutations. However, there are increasing concerns related to the transfection efficiency, cell viability, and maintenance of pluripotency provided by genome-editing techniques...
May 2018: Current Protocols in Stem Cell Biology
Ju Huang, Jing Li, Jun Zhou, Long Wang, Sihai Yang, Laurence D Hurst, Wen-Hsiung Li, Dacheng Tian
Repeated artificial selection of a complex trait facilitates the identification of genes underlying the trait, especially if multiple selected descendant lines are available. Here we developed a pedigree-based approach to identify genes underlying the Green Revolution (GR) phenotype. From a pedigree analysis, we selected 30 cultivars including the "miracle rice" IR8, a GR landmark, its ancestors and descendants, and also other related cultivars for identifying high-yield genes. Through sequencing of these genomes, we identified 28 ancestral chromosomal blocks that were maintained in all the high-yield cultivars under study...
July 23, 2018: Proceedings of the National Academy of Sciences of the United States of America
Lara Eisenbach, Dorothee Janßen, Matthias A Ehrmann, Rudi F Vogel
Lactobacillus (L.) curvatus reaches high numbers in a variety of habitats, which suggests a high (genomic) diversity within this species. Empirically selected strains are used as starter cultures in sausage fermentation. Determinants for the assertiveness of a strain in this environment are assumed to be multifactorial. We used comparative genomics and in silico proteomics of 10 L. curvatus strains, which were representative of its genetic and physiological biodiversity, to possibly derive genetic determinants for strain or group specific assertiveness in sausage fermentation...
July 4, 2018: International Journal of Food Microbiology
Viktória Hudacsek, Balázs Gyõrffy
Today, we have to investigate the effects of selected mutations and molecular alterations - this is made possible by the new genome editing technologies. In these, either a section of the DNA is deleted or moved, or a targeted mutation is introduced into the cell. Here, we describe in detail the CRISPR-Cas9 genome editing method. The technology is based on a feature used by bacteria to combat recurrent viral infections. At present, commencing a set of modifications, the method is functional and can be applied in eukaryotic cells as well...
July 20, 2018: Magyar Onkologia
Emily A Stout, Rosemary Sanozky-Dawes, Yong Jun Goh, Alexandra B Crawley, Todd R Klaenhammer, Rodolphe Barrangou
Lactobacillus gasseri is a human commensal which carries CRISPR-Cas, an adaptive immune system that protects the cell from invasive mobile genetic elements (MGEs). However, MGEs occasionally escape CRISPR targeting due to DNA mutations that occur in sequences involved in CRISPR interference. To better understand CRISPR escape processes, a plasmid interference assay was used to screen for mutants that escape CRISPR-Cas targeting. Plasmids containing a target sequence and a protospacer adjacent motif (PAM) were transformed for targeting by the native CRISPR-Cas system...
July 19, 2018: Microbiology
Anh D Ha, Dee R Denver
Bacteria of the genus Pseudomonas are genetically diverse and ubiquitous in the environment. Like other bacteria, those of the genus Pseudomonas are susceptible to bacteriophages which can significantly affect their host in many ways, ranging from cell lysis to major changes in morphology and virulence. Insights into phage genomes, evolution, and functional relationships with their hosts have the potential to contribute to a broader understanding of Pseudomonas biology, and the development of novel phage therapy strategies...
2018: Frontiers in Microbiology
Mrinal K Sarkar, Grace A Hile, Lam C Tsoi, Xianying Xing, Jianhua Liu, Yun Liang, Celine C Berthier, William R Swindell, Matthew T Patrick, Shuai Shao, Pei-Suen Tsou, Ranjitha Uppala, Maria A Beamer, Anshika Srivastava, Stephanie L Bielas, Paul W Harms, Spiro Getsios, James T Elder, John J Voorhees, Johann E Gudjonsson, J Michelle Kahlenberg
OBJECTIVE: Skin inflammation and photosensitivity are common in patients with cutaneous lupus erythematosus (CLE) and systemic lupus erythematosus (SLE), yet little is known about the mechanisms that regulate these traits. Here we investigate the role of interferon kappa (IFN-κ) in regulation of type I interferon (IFN) and photosensitive responses and examine its dysregulation in lupus skin. METHODS: mRNA expression of type I IFN genes was analysed from microarray data of CLE lesions and healthy control skin...
July 18, 2018: Annals of the Rheumatic Diseases
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