CRISPR-Cas9

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CRISPR gene editing

CRISPR gene editing (; pronounced like "crisper"; an abbreviation for "clustered regularly interspaced short palindromic repeats") 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 or new ones added in vivo. The technique is considered highly significant in biotechnology and medicine as it enables in vivo genome editing and is considered exceptionally precise, cost-effective, and efficient. It can be used in the creation of new medicines, agricultural products, and genetically modified organisms, or as a means of controlling pathogens and pests. It also offers potential in the treatment of inherited genetic diseases as well as diseases arising from somatic mutations, such as cancer. However, its use in human germline genetic modification is highly controversial. The development of this technique earned Jennifer Doudna and Emmanuelle Charpentier the Nobel Prize in Chemistry in 2020. The third researcher group that shared the Kavli Prize for the same discovery, led by Virginijus Šikšnys, was not awarded the Nobel Prize. Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted DNA sequence to introduce the modification by one of two methods. Knock-in mutations, facilitated via homology-directed repair (HDR), are the traditional pathway of targeted genomic editing approaches. This allows for the introduction of targeted DNA damage and repair. HDR employs the use of similar DNA sequences to drive the repair of the break via the incorporation of exogenous DNA to function as the repair template. This method relies on the periodic and isolated occurrence of DNA damage at the target site in order for the repair to commence. Knock-out mutations caused by CRISPR-Cas9 result from the repair of the double-stranded break by means of non-homologous end joining (NHEJ) or POLQ/polymerase theta-mediated end-joining (TMEJ). These end-joining pathways can often result in random deletions or insertions at the repair site, which may disrupt or alter gene functionality. Therefore, genomic engineering with CRISPR-Cas9 enables researchers to generate targeted random gene disruption. While genome editing in eukaryotic cells has been possible using various methods since the 1980s, the methods employed have proven to be inefficient and impractical for large-scale implementation. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing became possible. Cas9, derived from the bacterial species Streptococcus pyogenes, has facilitated targeted genomic modification in eukaryotic cells by enabling a reliable method of creating a targeted break at a specific location as designated by the crRNA and tracrRNA guide strands. Researchers can insert Cas9 and template RNA with ease in order to silence or cause point mutations at specific loci. This has proven invaluable for quick and efficient mapping of genomic models and biological processes associated with various genes in a variety of eukaryotes. Newly engineered variants of the Cas9 nuclease that significantly reduce off-target activity have been developed. CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing was the AAAS's choice for Breakthrough of the Year in 2015. Since 2015, CRISPR has been experimentally investigated on non-viable human embryos. In 2019, the first humans were born from genome-edited embryos using the CRISPR technique, as a result of the controversial He Jiankui affair. Several bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially the potential enabling of human eugenics. In 2023, the first drug making use of CRISPR gene editing, Exagamglogene autotemcel, sold under the brand name "Casgevy", was officially approved for use in the United Kingdom, to cure sickle-cell disease and beta thalassemia. On 2 December 2023, the Kingdom of Bahrain became the second country in the world to approve the use of Casgevy to treat sickle-cell anemia and beta thalassemia. On December 8, 2023, Casgevy received approval for use in the United States by the Food and Drug Administration.

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• crispr

  CRISPR gene editing is a revolutionary technology that allows for precise, targeted modifications to the DNA of living organisms. Developed from a natural defense mechanism found in bacteria, CRISPR-Cas9 is the most commonly used system. Gene editing with CRISPR-Cas9 involves a Cas9 nuclease and an engineered guide RNA, which come together to allow for the precise "cutting" of one or both strands of DNA at specific locations within the genome. It makes use of the cell's natural DNA repair [...] A simpler CRISPR system from S pyogenes uses Cas9, an endonuclease functioning with two small RNAs—crRNA and tracrRNA—to form a four-component complex. In 2012, Doudna and Charpentier simplified this into a two-component system by fusing the RNAs into a single-guide RNA, enabling Cas9 to target and cut specific DNA sequences—a breakthrough that earned them the 2020 Nobel Prize in Chemistry. Parallel work showed the S. thermophilus Cas9 could be similarly reprogrammed by altering the crRNA

• CRISPR gene editing - Wikipedia

  The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a gene-editing technology that can induce double-strand breaks (DSBs) anywhere guide ribonucleic acids (gRNA) can bind with the protospacer adjacent motif (PAM) sequence. Single-strand nicks can also be induced by Cas9 active-site mutants, also known as Cas9 nickases. By simply changing the sequence of gRNA, the Cas9-endonuclease can be delivered to a gene of interest and induce DSBs. The efficiency of [...] CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing was the AAAS's choice for Breakthrough of the Year in 2015. Since 2015, CRISPR has been experimentally investigated on non-viable human embryos. In 2019, the first humans were born from genome-edited embryos using the CRISPR technique, as a result of the controversial He Jiankui affair. Several bioethical concerns have been raised about the prospect of using CRISPR [...] In 2012, the research team, led by professor Jennifer Doudna (University of California, Berkeley) and professor Emmanuelle Charpentier (Umeå University), was the first group of individuals to identify, disclose, and file a patent application for the CRISPR-Cas9 system needed to edit DNA. They also published their finding that CRISPR-Cas9 could be programmed with RNA to edit genomic DNA, now considered one of the most significant discoveries in the history of biology.[citation needed]

• CRISPR-Cas Gene Editing Teaching Resources - Bio-Rad

  One of the most exciting recent developments in genetic engineering is CRISPR-Cas9 (CRISPR) gene editing. CRISPR technology allows scientists to edit genes and manipulate gene expression within living organisms. This allows the use of CRISPR gene editing in a far-reaching range of applications from basic research to the development of novel therapies and other biotechnology products. [...] The CRISPR-Cas9 system uses modified components of the bacterial CRISPR system to direct target-specific cutting of double-stranded DNA. DNA repair mechanisms then take over to fix the break in a manner that modifies the genetic sequence that has been cut. ### Step 1: Cutting the DNA Visit our YouTube CRISPR Gene Editing playlist for videos that depict the CRISPR-Cas9 method for genome editing. [...] The CRISPR-Cas9 microbial defense system. 1. The Cas1-Cas2 enzymes of the microbe recognize and cut out a segment of foreign DNA. 2. The Cas1-Cas2 enzymes insert the DNA segment into the CRISPR region of the bacterial genome as a spacer. 3. A spacer sequence is transcribed and then linked to a Cas9 protein. 4. Upon reinfection by the same invader, the CRISPR-Cas9 complex can recognize the foreign DNA sequence and cut it to prevent complete reinfection. ## CRISPR-Cas9 — A Gene Editing System

• CRISPR/Cas9 therapeutics: progress and prospects - Nature

  CRISPR/ Cas9 is a highly effective gene-editing tool that is widely used in the scientific community.6.") The CRISPR/Cas9 system evolved naturally in bacteria and archaea as a defense mechanism against phage infection and plasmid transfer.7."),8.") Bacteria or archaea acquire a segment of their DNA sequence to insert into the CRISPR spacer region when first infiltrated by an exogenous phage or plasmid. If reinfected with homologous DNA, the bacterium will initiate transcription of the CRISPR [...] CRISPR gene-editing technology facilitates gene editing in eukaryotic cells. Researchers have studied the mechanism of action of Cas9 and have obtained Cas9 variants with different functions and some other derivative gene-editing tools through special modifications and have discovered other Cas proteins in the Cas9 family, enriching the types of genes that can be edited using CRISPR technology. Researchers have developed some vectors to assist in transport and safely deliver the CRISPR system [...] The CRISPR/Cas family of proteins is divided into two categories based on genomic and protein structure information, and the best-known protein Cas9 is among the Class II CRISPR/Cas systems.49 protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 1, e60 (2005)."),50.") Class I is characterized by a large Cas9 protein complex that shears the DNA strand, while Class II requires only a single shearing protein. Cas9 is characterized by the presence of

• Questions and Answers about CRISPR

  A: CRISPR-Cas9 is proving to be an efficient and customizable alternative to other existing genome editing tools. Since the CRISPR-Cas9 system itself is capable of cutting DNA strands, CRISPRs do not need to be paired with separate cleaving enzymes as other tools do. They can also easily be matched with tailor-made “guide” RNA (gRNA) sequences designed to lead them to their DNA targets. Tens of thousands of such gRNA sequences have already been created and are available to the research [...] A: “CRISPR” (pronounced “crisper”) stands for Clustered Regularly Interspaced Short Palindromic Repeats, which are the hallmark of a bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology. In the field of genome engineering, the term “CRISPR” or “CRISPR-Cas9” is often used loosely to refer to the various CRISPR-Cas9 and -CPF1, (and other) systems that can be programmed to target specific stretches of genetic code and to edit DNA at precise locations, as well as [...] Research also suggests that CRISPR-Cas9 can be used to target and modify “typos” in the three-billion-letter sequence of the human genome in an effort to treat genetic disease. An artist's depiction of the CRISPR system in action. Illustration by Stephen Dixon Q: How does CRISPR-Cas9 compare to other genome editing tools?