Tag: CRISPOR

1. Introduction Staphylococcus aureus [1], one of the top five foodborne pathogens, has a common and strong aggressiveness in humans and can secrete multiple toxic proteins (Pathogenic enterotoxins, Hemolysin, PVL) [1,2] which can cause bacteraemia, endocarditis, meningitis, toxic shock syndrome, pneumonia and other dangerous infectious diseases [3]. Moreover, the worldwide…

Cell culture The human endometrial carcinoma HEC-1-B cells were cultured in the modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% (v/v) CO2 incubator. The human embryonic kidney HEK293T cells were cultured in the Dulbecco’s modified Eagle’s medium (DMEM) supplemented…

The cultivated strawberry (Fragaria × ananassa, Duch.) is one of the Rosaceae family renowned for its flavor and health benefits, making it an important agricultural commodity. However, its soft texture leads to a brief shelf life and consequent economic losses. Current breeding aims to enhance firmness without compromising quality, but…

Adams, R. H. & Eichmann, A. Axon guidance molecules in vascular patterning. Cold Spring Harb. Perspect. Biol. 2, a001875 (2010). Article  PubMed  PubMed Central  Google Scholar  Fish, J. E. & Wythe, J. D. The molecular regulation of arteriovenous specification and maintenance. Dev. Dyn. 244, 391–409 (2015). Article  CAS  PubMed  Google…

Susceptibility to Botrytis in wild type and FaPG1 edited line #13. Credit: Horticulture Research The cultivated strawberry (Fragaria × ananassa, Duch.) is one of the Rosaceae family renowned for its flavor and health benefits, making it an important agricultural commodity. However, its soft texture leads to a brief shelf life…

Rawla, P., Sunkara, T. & Gaduputi, V. Epidemiology of pancreatic cancer: Global trends, etiology and risk factors. World J. Oncol. 10, 10–27 (2019). Article  PubMed  PubMed Central  Google Scholar  Lambert, A. et al. An update on treatment options for pancreatic adenocarcinoma. Ther. Adv. Med. Oncol. 11, 1758835919875568 (2019). Article  PubMed …

Cloning of CRISPR/sgRNA lentiviral constructs with fluorescent selection markers A tetracycline-inducible promoter (TRE3G) was used to control the expression of S. aureus dCas9 in a lentiviral vector. To facilitate selection of cells by FACS, pHR:TRE3G-SadCas9-2xKRAB-p2a-tdTomato (Addgene ID #209298) was subcloned from a pHR:TRE3G-SadCas9-2xKRAB-p2a-zeo (A gift from Professor Stanley Qi), where zeocin…

Introduction The KCNQ (potassium voltage-gated channel subfamily Q) family of ion channels encode potassium transporters (1). KCNQ proteins typically repolarise the plasma membrane of a cell after depolarisation by allowing the export of potassium ions, and are therefore involved in wide-ranging biological functions including cardiac action potentials (2), neural excitability…

Klein, L., Kyewski, B., Allen, P. M. & Hogquist, K. A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don’t see). Nat. Rev. Immunol. 14, 377–391 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  Bornstein, C. et al. Single-cell mapping of the thymic stroma identifies…

Sleeping Beauty transgene overexpression Bidirectional Sleeping Beauty plasmids encoding the codon-optimized cDNA of the gene of interest (FLT3, KIT, CD123) or their mutated variants were cloned downstream of an EEF1A1 promoter. The same construct included a reporter cassette comprising a fluorescent protein (mCherry or mTagBFP2 for FLT3 and KIT, respectively)…

Introduction Immune checkpoint inhibitors (ICIs) have revolutionized the treatment pattern of various cancers, including melanoma and non-small cell lung cancer (NSCLC).1–3 However, there are still a considerable proportion (40%–60%) of patients who do not respond or do not achieve durable responses to these drugs.4 Thus, it is urgent clinical need…

Isolation of PFFs PFFs were isolated from the 30-day-old fetuses of a Large White pig purchased from Fujian Yongcheng Agricultrual & Animal Husbandry Science and Technology Group Co., Ltd (Jiangxi Province, China). Briefly, the sow was euthanized by intravenous injection of potassium chloride solution (100 mg/kg) under anesthesia by an intramuscular…

Compston, J. E., McClung, M. R. & Leslie, W. D. Osteoporosis. Lancet 393, 364–376 (2019). Article  CAS  PubMed  Google Scholar  Jha, S., Wang, Z., Laucis, N. & Bhattacharyya, T. Trends in media reports, oral bisphosphonate prescriptions, and hip fractures 1996–2012: an ecological analysis. J. Bone Miner. Res. 30, 2179–2187 (2015)….

Luo, Y. et al. New developments on the Encyclopedia of DNA Elements (ENCODE) data portal. Nucleic Acids Res. 48, D882–D889 (2020). Article  CAS  PubMed  Google Scholar  Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016). Article  CAS  PubMed …

Primers, plasmids, bacterial strains, and growth conditions Primers, plasmids, and strains used in this study are listed in Tables S1–S3, respectively. Escherichia coli strains MG1655, DH10B, MDS42, and their derivates were cultured in LB medium at 37 °C, with shaking at 220 rpm. All the recombinants containing the temperature-sensitive pSC101-derived plasmid were cultured…

Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 1987;169(12):5429–33. Article  CAS  PubMed  PubMed Central  Google Scholar  Bhaya D, Davison M, Barrangou R. CRISPR-Cas systems in…

Hill SK, Reilly JL, Keefe RS, Gold JM, Bishop JR, Gershon ES, Tamminga CA, Pearlson GD, Keshavan MS, Sweeney JA. Neuropsychological impairments in schizophrenia and psychotic bipolar disorder: findings from the Bipolar-Schizophrenia Network on Intermediate Phenotypes (B-SNIP) study. Am J Psychiatry. 2013;170(11):1275–84. PubMed  PubMed Central  Google Scholar  Penzes P, Cahill…

Statement on ethical considerations All animal work was approved and permitted by the Local Ethical Committee on Animal Experiments and conducted according to the Guidelines for Animal Experimentation recommendations (ARRIVE guidelines). In particular, mouse work related to C57BL/6NCrl mice was approved and permitted by the Institute of Molecular Genetics of…

Laurenti, E. & Gottgens, B. From haematopoietic stem cells to complex differentiation landscapes. Nature (London) 553, 418–426 (2018). ADS  CAS  PubMed  Google Scholar  Miller, I. J. & Bieker, J. J. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Krüppel family…

Cell culture We cultured mESCs in t2iL medium containing Dulbecco’s modified eagle medium (DMEM, Nacalai Tesque), 2 mM Glutamax (Nacalai Tesque), 1× non-essential amino acids (Nacalai Tesque), 1 mM sodium pyruvate (Nacalai Tesque), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin (P/S) (Nacalai Tesque), 0.1 mM 2-mercaptoethanol (Sigma) and 15% fetal bovine serum (FBS) (Gibco), supplemented with…

Cell culture IMR90 (CCL-186) and WI38 (AG06814-N) fibroblasts were purchased from ATCC and the Coriell Institute for Medical Research, respectively. IMR90 and WI38 fibroblasts were grown under 7.5% CO2 and 3% O2 in GlutaMax-DMEM (Gibco, 10569-010) supplemented with 0.1 mM non-essential amino acids (Corning, 25-025-Cl) and 15% fetal bovine serum (VWR/Seradigm,…

Generation, genotyping, and off-target analysis of Trem2 H157Y knock-in mice Trem2 H157Y knock-in mice were generated via CRISPR/Cas9 by the Hope Center Transgenic Vectors Core of the Washington University [25]. CRISPR gRNAs for in vitro testing were identified using CRISPOR (crispor.tefor.net/) and synthesized as gBlocks (Integrated DNA Technologies, IDT) with…

Here we provide a tool that directly selects biallelically edited knock-ins using cell-surface displays, thereby eliminating the need for extensive clonal verification. By doing so SNEAK PEEC greatly reduces the considerable effort currently expended to obtain biallelically edited clones. The ability to generate a clonal population with multiple edits is…

Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020). Article  CAS  Google Scholar  Clement, K., Hsu, J. Y., Canver, M. C., Joung, J. K. & Pinello, L. Technologies and computational analysis strategies for…

Cell isolation, reprogramming and culture Approval was obtained for human cell and tissue sample collection and genetic reprogramming from the Institutional Review Board (protocols 19–252, 18–243, 21–060, 19–284 and 415-E/1776/4-2014, Ethics Committee of the province of Salzburg). Adult samples were collected in accordance with the Declaration of Helsinki after written…

Bartel, D. P. MicroRNAs: Target recognition and regulatory functions. Cell 136, 215–233 (2009). CAS  Article  Google Scholar  Bassett, A. R. et al. Understanding functional miRNA–target interactions in vivo by site-specific genome engineering. Nat. Commun. 5, 4640 (2014). ADS  CAS  Article  Google Scholar  Moore, M. J. et al. miRNA–target chimeras reveal…

1. Dunbar, C. E. et al. Gene therapy comes of age. Science 359, eaan4672 (2018). PubMed Google Scholar  2. Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov. 18, 358–378 (2019). CAS PubMed PubMed Central Google Scholar  3. Eid, A.,…