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Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.
Functional genomics is a field of molecular biology that attempts to describe gene functions and interactions. Functional genomics make use of the vast data generated by genomic and transcriptomic projects. Functional genomics focuses on the dynamic aspects such as gene transcription, translation, regulation of gene expression and protein–protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structures. A key characteristic of functional genomics studies is their genome-wide approach to these questions, generally involving high-throughput methods rather than a more traditional “gene-by-gene” approach.
A designer baby is a baby whose genetic makeup has been selected or altered, often to include a particular gene or to remove genes associated with a disease. This process usually involves analysing a wide range of human embryos to identify genes associated with particular diseases and characteristics, and selecting embryos that have the desired genetic makeup; a process known as preimplantation genetic diagnosis. Other potential methods by which a baby's genetic information can be altered involve directly editing the genome before birth. This process is not routinely performed and only one instance of this is known to have occurred as of 2019, where Chinese twins Lulu and Nana were edited as embryos, causing widespread criticism.
CRISPR 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 defense system of prokaryotes.
Guide RNAs are the RNAs that guide the insertion or deletion of uridine residues into mitochondrial mRNAs in kinetoplastid protists in a process known as RNA editing.
Breast cancer anti-estrogen resistance protein 1 is a protein that in humans is encoded by the BCAR1 gene.
Maternal embryonic leucine zipper kinase (MELK) is an enzyme that in humans is encoded by the MELK gene. MELK is a serine/threonine kinase belonging to the family of AMPK/Snf1 protein kinases. MELK was first identified present as maternal mRNA in mouse embryos. MELK expression is elevated in a number of cancers and is an active research target for pharmacological inhibition.
Cas9 is a 160 kilo dalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids and which is heavily utilized in genetic engineering applications. Its main function is to cut DNA and therefore it can alter a cell's genome.
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).
Epigenome editing or Epigenome engineering is a type of genetic engineering in which the epigenome is modified at specific sites using engineered molecules targeted to those sites. Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question.
A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and hence is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.
CRISPR-Cas design tools are computer software platforms and bioinformatics tools used to facilitate the design of guide RNAs (gRNAs) for use with the CRISPR/Cas gene editing system.
CRISPR-associated protein 1 (cas1) is one of the two universally conserved proteins found in the CRISPR prokaryotic immune defense system. Cas1 is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments. Cas1 forms a stable complex with the other universally conserved CRISPR-associated protein, cas2, which is essential to spacer acquisition for CRISPR systems.
Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cpf1 is a DNA-editing technology analogous to the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. CRISPR/Cpf1 could have multiple applications, including treatment of genetic illnesses and degenerative conditions.
CRISPR activation (CRISPRa) is one type of CRISPR tool that use modified versions of dCas9, a mutation of Cas9 without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs). Like a standard CRISPR-Cas9 system, dCas9 activation systems rely on similar components such as Cas9 variants for modulation or modification of genes, gRNAs to guide Cas9 to intended targets, and vectors for introduction into cells. However, while a standard CRISPR-Cas9 system relies on creating breaks in DNA through the endonuclease activity of Cas9 and then manipulating DNA Repair mechanisms for gene editing, dCas9 activation systems are modified and employ 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.
Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA (“target”), which they cleave, creating a double-stranded chromosomal break (DSB) that summons the cell's DNA repair mechanisms and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations.
CRISPR gene editing is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.
Locus Biosciences is a preclinical-stage pharmaceutical company, founded in 2015 and based in Research Triangle Park, North Carolina which aims to develop phage therapies based on CRISPR–Cas3 gene editing technology, as opposed to the more commonly used CRISPR-Case9, delivered by engineered bacteriophages, under the trademark crPhage. The intended therapeutic target of crPhase is antibiotic-resistant bacterial infections.
Anti-CRISPR is a group of proteins found in phages, that inhibit the normal activity of CRISPR-Cas, the immune system of certain bacteria. CRISPR consists of genomic sequences that can be found in prokaryotic organisms, that come from bacteriophages that infected the bacteria beforehand, and are used to defend the cell from further viral attacks. Anti-CRISPR results from an evolutionary process occurred in phages in order to avoid having their genomes destroyed by the prokaryotic cells that they will infect.
Genome-wide CRISPR-Cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genome-wide scale and studying the resulting phenotypic alterations. The approach utilises the CRISPR-Cas9 gene editing system, coupled with libraries of single guide RNAs (sgRNAs), which are designed to target every gene in the genome. Over recent years, the genome-wide CRISPR screen has emerged as a powerful tool for performing large-scale loss-of-function screens, with low noise, high knockout efficiency and minimal off-target effects.
References
- 1 2 Lee H, Dhingra Y, Sashital DG (April 2019). "The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation". eLife. 8. doi: 10.7554/eLife.44248 . PMID 31021314.
- 1 2 Lee H, Zhou Y, Taylor DW, Sashital DG (April 2018). "Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays". Molecular Cell. 70 (1): 48–59.e5. doi: 10.1016/j.molcel.2018.03.003 . PMID 29602742.
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