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CRISPRi
CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim, Adam Arkin, Jonathan Weissman, and Jennifer Doudna. Sequence-specific activation of gene expression refers to CRISPR activation (CRISPRa). Based on the bacterial genetic immune system - CRISPR (clustered regularly interspaced short palindromic repeats) pathway, the technique provides a complementary approach to RNA interference. The difference between CRISPRi and RNAi, though, is that CRISPRi regulates gene expression primarily on the transcriptional level, while RNAi controls genes on the mRNA level. Background Many bacteria and most archaea have an adaptive immune system which incorporates CRISPR RNA (crRNA) and CRISPR-associated (cas) genes. The CRISPR interference (CRISPRi) technique was first reported by Lei S. Qi an ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Perturb-seq
Perturb-seq (also known as CRISP-seq and CROP-seq) refers to a high-throughput method of performing single cell RNA sequencing (scRNA-seq) on pooled genetic perturbation screens. Perturb-seq combines multiplexed CRISPR mediated gene inactivations with single cell RNA sequencing to assess comprehensive gene expression phenotypes for each perturbation. Inferring a gene’s function by applying genetic perturbations to knock down or knock out a gene and studying the resulting phenotype is known as reverse genetics. Perturb-seq is a reverse genetics approach that allows for the investigation of phenotypes at the level of the transcriptome, to elucidate gene functions in many cells, in a massively parallel fashion. The Perturb-seq protocol uses CRISPR technology to inactivate specific genes and DNA barcoding of each guide RNA to allow for all perturbations to be pooled together and later deconvoluted, with assignment of each phenotype to a specific guide RNA. Droplet-based microflui ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Stanley Qi
Lei "Stanley" Qi (; born August 17, 1983) is an assistant professor in the Department of Bioengineering, and the Department of Chemical and Systems Biology at Stanford University. Qi led the development of the first catalytically dead Cas9 lacking endonuclease activity (dCas9), which is the basis for CRISPR interference (CRISPRi). His laboratory subsequently developed CRISPR-Genome Organization (CRISPR-GO). Qi is a co-inventor of the University of California patent on the CRISPR gene-editing technology. Early life and education Qi obtained his B.S. in physics and math from Tsinghua University, China, Master in physics from UC Berkeley The University of California, Berkeley (UC Berkeley, Berkeley, Cal, or California) is a public university, public land-grant university, land-grant research university in Berkeley, California. Established in 1868 as the University of Californi ..., and PhD in bioengineering from UC Berkeley. During his PhD work at Berkeley, he studied synthe ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
CRISPR Sterics
CRISPR () (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti-phage) defense system of prokaryotes and provide a form of acquired immunity. CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. This editin ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Krüppel Associated Box
The Krüppel associated box (KRAB) domain is a category of transcriptional repression domains present in approximately 400 human zinc finger protein-based transcription factors (KRAB zinc finger proteins). The KRAB domain typically consists of about 75 amino acid residues, while the minimal repression module is approximately 45 amino acid residues. It is predicted to function through protein-protein interactions via two amphipathic helices. The most prominent interacting protein is called TRIM28 initially visualized as SMP1, cloned as KAP1 and TIF1-beta. Substitutions for the conserved residues abolish repression. Over 10 independently encoded KRAB domains have been shown to be effective repressors of transcription, suggesting this activity to be a common property of the domain. KRAB domains can be fused with dCas9 CRISPR tools to form even stronger repressors. Evolution The KRAB domain had initially been identified in 1988 as a periodic array of leucine residues separated b ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
DCas9 Activation System
CRISPR activation (CRISPRa) is a type of CRISPR tool that uses modified versions of CRISPR effectors without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs). Like for CRISPR interference, the CRISPR effector is guided to the target by a complementary guide RNA. However, CRISPR activation systems are fused to transcriptional activators to increase expression of genes of interest. Such systems are usable for many purposes including but not limited to, genetic screens and overexpression of proteins of interest. The most commonly-used effector is based on Cas9 (from Type II systems), but other effectors like Cas12a (Type V) have been used as well. Components dCas9 Cas9 Endonuclease Dead, also known as dead Cas9 or dCas9, is a mutant form of Cas9 whose endonuclease activity is removed through point mutations in its endonuclease domains. Similar to its unmutated form, dCas9 is used in CRISPR systems along with gRNAs to target specific ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
CRISPR
CRISPR () (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti-phage) defense system of prokaryotes and provide a form of acquired immunity. CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. This editing ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Wendell Lim
Wendell Lim Ph.D. is the Byer's Distinguished Professor of Cellular and Molecular Pharmacology at the University of California, San Francisco. He is the Director of the UCSF Cell Design Institute. He earned his A.B. in Chemistry from Harvard University working with Jeremy Knowles on enzyme evolutionary optimization. He obtained his Ph.D in biochemistry and biophysics from Massachusetts Institute of Technology under the guidance of Bob Sauer using genetic and biophysical approaches to understand the role of hydrophobic core interactions in protein folding. He then did his postdoctoral work with Frederic Richards at Yale University on the structure of protein interaction domains. Lim's work has focused on cell signaling, synthetic biology, and cell engineering, particularly in immune cells. Research career Modular Signaling Proteins Lim’s research has focused on mechanisms of cell signaling – how cells sense their environment and process this information to make comple ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Induced Pluripotent Stem Cell
Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka's lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes (named Myc, Oct3/4, Sox2 and Klf4), collectively known as Yamanaka factors, encoding transcription factors could convert somatic cells into pluripotent stem cells. He was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent." Pluripotent stem cells hold promise in the field of regenerative medicine. Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic, and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease. The most well-known type of pluripotent stem cell is the ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Horizontal Gene Transfer
Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between Unicellular organism, unicellular and/or multicellular organisms other than by the ("vertical") transmission of DNA from parent to offspring (reproduction). HGT is an important factor in the evolution of many organisms. HGT is influencing scientific understanding of higher order evolution while more significantly shifting perspectives on bacterial evolution. Horizontal gene transfer is the primary mechanism for the spread of antibiotic resistance in bacteria, and plays an important role in the evolution of bacteria that can degrade novel compounds such as human-created Bactericide, pesticides and in the evolution, maintenance, and transmission of virulence. It often involves Temperateness (virology), temperate bacteriophages and plasmids. Genes responsible for antibiotic resistance in one species of bacteria can be transferred to another species of bacteria through various m ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Reprogramming
In biology, reprogramming refers to erasure and remodeling of epigenetic marks, such as DNA methylation, during mammalian development or in cell culture. Such control is also often associated with alternative covalent modifications of histones. Reprogrammings that are both large scale (10% to 100% of epigenetic marks) and rapid (hours to a few days) occur at three life stages of mammals. Almost 100% of epigenetic marks are reprogrammed in two short periods early in development after fertilization of an ovum by a sperm. In addition, almost 10% of DNA methylations in neurons of the hippocampus can be rapidly altered during formation of a strong fear memory. After fertilization in mammals, DNA methylation patterns are largely erased and then re-established during early embryonic development. Almost all of the methylations from the parents are erased, first during early embryogenesis, and again in gametogenesis, with demethylation and remethylation occurring each time. Demethyla ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Green Fluorescent Protein
The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea victoria'' and is sometimes called ''avGFP''. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets. The GFP from ''A. victoria'' has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. The GFP from the sea pansy (''Renilla reniformis'') has a single major excitation peak at 498 nm. GFP makes for an excellent tool in many forms of biology due to its ability to form an internal chromophore without requiring any accessory cofactors, gene products, or enzymes / substrates other than mo ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Muller's Morphs
Hermann J. Muller (1890–1967), who was a 1946 Nobel Prize winner, coined the terms amorph, hypomorph, hypermorph, antimorph and neomorph to classify mutations based on their behaviour in various genetic situations, as well as gene interaction between themselves.Muller, H. J. 1932. Further studies on the nature and causes of gene mutations. ''Proceedings of the 6th International Congress of Genetics'', pp. 213–255. These classifications are still widely used in ''Drosophila'' genetics to describe mutations. For a more general description of mutations, see mutation, and for a discussion of allele interactions, see dominance relationship. ''Key: In the following sections, alleles are referred to as +=wildtype, m=mutant, Df=gene deletion, Dp=gene duplication. Phenotypes are compared with '>', meaning 'phenotype is more severe than Loss of function Amorph Amorphic describes a mutation that causes complete loss of gene function. Amorph is sometimes used interchangeably ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
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