Tag: CRISPOR
CRISPR-created strawberries that stay firmer and fresher longer could revolutionize fruit industry
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…
Mutation of key signaling regulators of cerebrovascular development in vein of Galen malformations
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…
CRISPR/Cas9 editing enhances fruit firmness and extends shelf life
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…
CRISPR/Cas9-mediated knock-in of BRCA1/2 mutations restores response to olaparib in pancreatic cancer cell lines
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 …
Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson’s disease
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…
KCNQ potassium channels modulate Wnt activity in gastro-oesophageal adenocarcinomas
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…
Thymic mimetic cells function beyond self-tolerance
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…
Epitope editing enables targeted immunotherapy of acute myeloid leukaemia
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)…
Inhibition of tumor intrinsic BANF1 activates antitumor immune responses via cGAS-STING and enhances the efficacy of PD-1 blockade
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…
Efficient delivery of a large-size Cas9-EGFP vector in porcine fetal fibroblasts using a Lonza 4D-Nucleofector system | BMC Biotechnology
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…
Converging evidence from exome sequencing and common variants implicates target genes for osteoporosis
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)….
Dynamic network-guided CRISPRi screen identifies CTCF-loop-constrained nonlinear enhancer gene regulatory activity during cell state transitions
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 …
Insertion sequence transposition inactivates CRISPR-Cas immunity
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…
Targeting miRNA by CRISPR/Cas in cancer: advantages and challenges | Military Medical Research
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…
Identification of a psychiatric risk gene NISCH at 3p21.1 GWAS locus mediating dendritic spine morphogenesis and cognitive function | BMC Medicine
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…
A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology
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…
Identification of a genomic DNA sequence that quantitatively modulates KLF1 transcription factor expression in differentiating human hematopoietic cells
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…
Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs
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…
Telomere-to-mitochondria signalling by ZBP1 mediates replicative crisis
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,…
Trem2 H157Y increases soluble TREM2 production and reduces amyloid pathology | Molecular Neurodegeneration
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…
Rapid clonal identification of biallelic CRISPR/Cas9 knock-ins using SNEAK PEEC
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…
Human genetic diversity alters off-target outcomes of therapeutic gene editing
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…
Extra-hematopoietic immunomodulatory role of the guanine-exchange factor DOCK2
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…
CRISPRi for specific inhibition of miRNA clusters and miRNAs with high sequence homology
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…
dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy
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.,…
