Tag: BsmBI

ESRP1 controls biogenesis and function of a large abundant multiexon circRNA | Nucleic Acids Research

Abstract While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2–27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal…

Continue Reading ESRP1 controls biogenesis and function of a large abundant multiexon circRNA | Nucleic Acids Research

Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer

Mice The research conducted in this study complied with all of the relevant ethical regulations. The animal protocols were approved by and performed in accordance with the Institutional Animal Care and Use Committee of St. Jude Children’s Research Hospital. C57BL/6, OT-I50, pmel51 and Rosa26-Cas9 knock-in52 mice were purchased from The…

Continue Reading Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer

Transcriptional linkage analysis with in vivo AAV-Perturb-seq

Experimental procedures Plasmid design and cloning AAV genome plasmids (Fig. 1a and Extended Data Figs. 1a,g,h and 5a) were based on Addgene plasmid 60231 (ref. 12). To achieve widespread transgene expression, the hSyn promoter was replaced by the ubiquitous CBh promoter (pAS088). For the triple-colour experiments (Extended Data Fig. 1a),…

Continue Reading Transcriptional linkage analysis with in vivo AAV-Perturb-seq

Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity

Mice All animal use was approved by the Department of Comparative Medicine at the Massachusetts Institute of Technology (MIT) and the Institutional Animal Care and Use Committee under protocol no. 0714-076-17. Mice were housed with a 12-h light/12-h dark cycle with temperatures in the range 20–22 °C and 30–70% humidity. KrasLSL-G12D…

Continue Reading Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity

AAV-mediated base-editing therapy ameliorates the disease phenotypes in a mouse model of retinitis pigmentosa

Ethics statement This study did not involve human data. All animal procedures were approved by the Institutional Review Board of Shanghai Jiao Tong University and were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research. Animals…

Continue Reading AAV-mediated base-editing therapy ameliorates the disease phenotypes in a mouse model of retinitis pigmentosa

Optimized minimal genome-wide human sgRNA library

sgRNA collection and enzyme cut site annotation All sgRNAs corresponding to potential NGG-containing target sites on the human (GRCh38/hg38) genome were calculated by a customized Perl script. The enzyme cut site was set at the 17 position from 5’ to 3’ of each sgRNA sequence. These sgRNAs have more than…

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Method for pig SLA-2 gene specific knockout through CRISPR-Cas9 and sgRNA for specially targeting SLA-2 gene – Eureka

[0090] Embodiment 2, constructing the sgRNA expression vector of SLA-2 gene [0091] 1. Synthesis of DNA Inserts [0092] (1) Synthesize the forward and reverse oligonucleotide sequences designed above [0093] The oligonucleotide sequence can be specifically synthesized by a commercial company (such as Invitrogen) according to the provided sequence. In this…

Continue Reading Method for pig SLA-2 gene specific knockout through CRISPR-Cas9 and sgRNA for specially targeting SLA-2 gene – Eureka

Addgene: AAV-EFS-SaKKHABE8e-bGH-U6-sgRNA-BsmBI

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications. For your Materials & Methods section: AAV-EFS-SaKKHABE8e-bGH-U6-sgRNA-BsmBI was a gift from David Liu (Addgene plasmid # 189923…

Continue Reading Addgene: AAV-EFS-SaKKHABE8e-bGH-U6-sgRNA-BsmBI

Multimodal perturbation analyses of cyclin-dependent kinases reveal a network of synthetic lethalities associated with cell-cycle regulation and transcriptional regulation

Phylogenetic tree construction Tree diagram showing relationships between CDK proteins was constructed from a multi-sequence alignment (MSA) using Geneious95. The “Geneious Aligner”, was used to generate the MSA, and the neighbor joining method was used to construct the tree. All default parameters were used except where otherwise indicated. Combinatorial CRISPR…

Continue Reading Multimodal perturbation analyses of cyclin-dependent kinases reveal a network of synthetic lethalities associated with cell-cycle regulation and transcriptional regulation

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…

Continue Reading Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs

SARS-CoV-2 restructures host chromatin architecture

Cell culture Human lung adenocarcinoma cells A549 expressing human ACE2 (A549-ACE2, NR-53821) were acquired from BEI Resources. They were maintained in DMEM/F-12 (1:1, Corning) medium supplemented with 10% FBS (GeneDepot) and blasticidin (100 μM). Normal A549 cells were purchased from ATCC (CCL-185) and cultured in DMEM/F-12 (1:1, Corning) supplemented with 10%…

Continue Reading SARS-CoV-2 restructures host chromatin architecture

Whole-genome doubling drives oncogenic loss of chromatin segregation

Cell culture hTERT-RPE-1 WT and hTERT RPE-1 TP53−/− (46, XX)27 cells were a gift from J. Korbel. The cells were grown in DMEM/F-12, GlutaMAX (10565018) supplemented with 10% FBS (Thermo Fisher Scientific, 10270106) and 1% antibiotic–antimycotic (Thermo Fisher Scientific, 15240062). CP-A (KR-42421) (47, XY) cells were purchased from the American Type…

Continue Reading Whole-genome doubling drives oncogenic loss of chromatin segregation

MacroH2A histone variants modulate enhancer activity to repress oncogenic programs and cellular reprogramming

Cell culture Normal Human Melanocytes (NHM) were cultured in Dermal Cell Basal Medium (ATCC) with the addition of 5 µg/ml Insulin, 50 µg/ml Ascorbic Acid, 6 mM L-Glutamine, 1.0 µM Epinephrine, 1.5 mM Calcium Chloride, Peptide Growth Factor and M8 Supplement. Dermal fibroblasts (DFs) were isolated from neonatal mice and iPS reprogramming was performed as…

Continue Reading MacroH2A histone variants modulate enhancer activity to repress oncogenic programs and cellular reprogramming