Crispr Cas9
CRISPR-Cas9 is a revolutionary gene-editing technology that allows scientists to precisely modify DNA within living organisms. It is derived from a natural defense mechanism found in bacteria, which uses RNA molecules and the Cas9 protein to target and cut specific DNA sequences. This technology has the potential to revolutionize fields such as medicine, agriculture, and biotechnology.
8 Key excerpts on "Crispr Cas9"
eBook - ePubHuman Genetics and Genomics
A Practical Guide
- Bahar Taneri, Esra Asilmaz, Türem Delikurt, Pembe Savas, Seniye Targen, Yagmur Esemen(Authors)
- 2020(Publication Date)
- Wiley-VCH(Publisher)
...11 Evolving Tools in Genome Editing: CRISPR-Cas Learning Outcomes Upon completing this practical the students will be able to understand the basic genome editing mechanisms; describe the CRISPR-Cas system; investigate the applications of CRISPR-Cas tool. Background Genome editing is a process that provides targeted modifications to the genome through various ways, including but not limited to addition and/or deletion in transcription factor binding sites, deletion in a promoter site, insertion of an inducible or constitutive promoter, disruption of a gene by knockout, point mutation in an exon, insertion of a new exon to make gene fusions, fusion of a reporter gene, and addition and/or deletion of a microRNA control element to 3′ untranslated region [ 1 ]. Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system has been introduced as a genome editing tool, which is based on the adaptive immunity response of bacteria and archaea [2]. In this chapter, the CRISPR-Cas system is briefly over-viewed. It should be noted that there are various detailed, cutting-edge reviews available on the topic. Living organisms have to cope with biotic and abiotic stress factors in nature. Several defense and regulatory mechanisms have evolved among living organisms in order to survive under unfavorable conditions. One such example is the defense system of the bacteria and archaea to cope with virus attacks. Some viruses, such as the bacteriophages, insert their genetic materials into the host genome. The virus fuses its genetic material into the host genome region, which contains the CRISPR locus. Viral DNA is inserted into the spacer regions flanked by the short palindromic repeat sequences, which produces heritable adaptive immunity in the host...
eBook - ePubGenetics 101
From Chromosomes and the Double Helix to Cloning and DNA Tests, Everything You Need to Know about Genes
- Beth Skwarecki(Author)
- 2018(Publication Date)
- Adams Media(Publisher)
...Bacteria fight them by chopping their DNA (or RNA) into pieces. The bacteria can copy a snippet of the virus’s sequence into their own chromosome, making a scrapbook of all the invaders they have defeated. The snippets in the scrapbook are separated by special repeats of DNA. Scientists didn’t realize at first why they were finding sections of bacterial DNA with repeats, then a unique sequence, then more repeats, and so on. They named this odd configuration “clustered regularly interspaced short palindromic repeats.” For short: CRISPR. Bacteria that had CRISPR sequences also had genes that made DNA-binding proteins, and these are the Cas, or CRISPR-associated genes. HOW CRISPR AND CAS9 EDIT DNA In the wild, the bacterium makes an RNA copy of one or more of its scrapbooked sequences from the CRISPR array, and a protein called Cas9 holds onto it. This is the guide RNA: the thing that Cas9 is programmed to search and destroy. When it finds a piece of DNA floating around in the cell, it cuts that DNA. Mission accomplished. Scientists figured out that they can get Cas9 to cut any spot in any genome, so long as they give it the right guide RNA so it knows what to look for. That makes this a programmable enzyme. Cutting DNA sounds like bad news, but don’t forget we have systems for repairing damaged DNA. If Cas9 just cuts, the cell can usually figure out how to put the remaining DNA back together. Or scientists can provide an extra piece of DNA for the repair enzymes to insert. And here’s the best part: this system can be used not just in bacteria but in any type of cell, including human cells. HOW SCIENTISTS ARE USING CRISPR/CAS9 An enzyme that can find a DNA sequence, cut it, and optionally insert any sequence that you want, is a very powerful tool...
eBook - ePubBiosafety and Bioethics in Biotechnology
Policy, Advocacy, and Capacity Building
- Sylvia Uzochukwu, Nwadiuto (Diuto) Esiobu, Arinze Stanley Okoli, Emeka Godfrey Nwoba, Ezebuiro Nwagbo Christpeace, Charles Oluwaseun Adetunji, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Sylvia Uzochukwu, Nwadiuto (Diuto) Esiobu, Arinze Stanley Okoli, Emeka Godfrey Nwoba, Ezebuiro Nwagbo Christpeace, Charles Oluwaseun Adetunji, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi(Authors)
- 2022(Publication Date)
- CRC Press(Publisher)
...In the immune system of bacteria and archae, the CRISPR/Cas system acts in an adaptive manner similar to the adaptive immune system of eukaryotes. The CRISPR/Cas system protects bacteria and archae from invading viruses and plasmids and retains memory of the invasion which is recruited to protect the host in subsequent invasion by the same virus or plasmid (Jinek et al. 2012). In bacteria, these defence systems rely on enzymes (Cas proteins) and small RNAs (CRISPR RNAs-crRNAs) for sequence-specific detection and destruction of the nucleic acids of viruses and plasmids (Jinek et al. 2012). This technology is now widely explored as a therapeutic strategy against infections (Bikard et al. 2014), various non-malignant and malignant diseases (van Diemen and Lebbink 2017), and in vaccine development and gene therapy (Schwank et al. 2013). In cancer treatment, clinical trials of CRISPR/Cas9-based therapy have been initiated (ClinicalTrials.gov 2017), and several preclinical studies involving CRISPR/Cas9-mediated correction of human genetic diseases are underway. Genome editing in crops can greatly speed up new trait development, and drive improvements in yield and pest and disease resistance as well as biofortification improvement and adaptation to climate change. Genome editing in animals has been used to increase disease resistance, to make livestock better adapted to farming or environmental conditions, to increase fertility and growth, and to improve animal welfare (Ricrock 2019). 2.2.3 Limitations of CRISPR/Cas9 Target specificity and off-target effects: due to the short sequence length of the gRNA (20 nt) and that of the PAM (3 nt), the gRNA-Cas9 complex often binds and mutates at non-specific loci which are similar, but not identical, in homology to target sites...
eBook - ePub- Vijai Singh, Pawan K. Dhar(Authors)
- 2020(Publication Date)
- Academic Press(Publisher)
...Application of CRISPR/Cas9 systems CRISPR/Cas9 technology initiated novel epoch of genetic engineering, which open an unprecedented opportunities for precise genome editing. Since it was first harnessed, Cas9 technology has been successfully used in medicine, agriculture and (synthetic) biotechnology for diverse applications. CRISPR/Cas9 technology has surpassed other previous prevalent genome editing method such as ZFN (Carroll, 2011) and TALEN (Chaudhary et al., 2016 ; Joung and Sander, 2013), due to its simplicity, inexpensive, specificity, easy to design and amenability to multiplexing (Wright et al., 2016 ; Doudna and Charpentier, 2014). Further, Cas9 was repurposed by induced a single mutations in its catalytic domain RuvC (D10A) and HNH (H840A) generate catalytically inactive also called dead Cas9 (dCas9). Dead Cas9 has no nuclease activity but it can bind to DNA. dCas9 can be coupled to transcriptional activation domain (VP16/VP64) or repression domain (KRAB/SID) to mediated transcription regulation (CRISPRa/CRISPRi) (Qi et al., 2013 ; Gilbert et al., 2013). CRISPRi is a more specific technology for gene silencing as compared to RNAi. dCas9 has also been used to modulate transcription state of specific genomic loci, assessing epigenetic state (Bikard et al., 2013 ; Hilton et al., 2015) Also, by fusing dCas9 with fluorescent marker like GFP (green fluorescent protein) has been used for labeling specific chromosomal loci, providing another method for live cell tracking (Chen et al., 2013). Another modification of Cas9 called base editor, a system in which dCas9 fused with cytidine deaminase (convert cytosine to uracil), which modify specific base in precise way (Kim et al., 2017a)...
eBook - ePubGenome Editing in Plants
Principles and Applications
- Om Prakash Gupta, Suhas Gorakh Karkute, Om Prakash Gupta, Suhas Gorakh Karkute(Authors)
- 2021(Publication Date)
- CRC Press(Publisher)
...Basically, in vivo CRISPR/Cas9 incorporates two domains: for instance, a protein (Cas9 nuclease) and a programmable single guide RNA (sgRNA). sgRNA is a unit of two RNAs: a developed CRISPR RNA (crRNA) and a partially corresponding trans-activating tracrRNA. A sequence of DNA can be targeted by Cas unit only when NGG protospacer-adjacent motifs, i.e. PAM sequences, are present. These essential components of this system have been discussed one by one in this chapter. We have also elaborately discussed the evolution of CRISPR/Cas as an adaptive defence mechanism in archaea and bacteria; the mechanism of CRISPR/Cas9-mediated genome editing in plants; and the comparison of CRISPR/Cas with other techniques of genome editing like meganucleases, ZFNs and TALENs, and also about the modified form of CRISPR, i.e. prime editing. 4.2 CRISPR: History and Adaptive Defence Mechanism in Bacteria and Archaea Immune system of animals and prokaryotes is supposed to be the most advanced system as a lot of pathways are triggered after the attack of any pathogen. Prokaryotes, much simpler than a eukaryotic system, also have developed an immune system to keep away bacteriophage or plasmid (Horvath and Barrangou, 2010). One of the systems is called CRISPR/Cas, which consists of non-repetitive spacer sequences unique in nature and adjacent to spacer are present 6–20 genes, which encode for Cas proteins (Lillestøl et al., 2006). The repeats found in each CRISPR locus have 23–47 numbers of base pairs and are highly conserved in nature. Horvath and Barrangou revealed that spacers are of 21–72 base pairs in length and composed of extrachromosomal elements. In 1987, some scientists cloned and sequenced iap genes in Escherichia coli. Later on, studies on iap genes reported repeats having 29 nucleotide base pairs, which were separated by non-repetitive spacer sequences (Nakata et al., 1989)...
eBook - ePub- Frank Thelen, Markus Schorn(Authors)
- 2020(Publication Date)
- Frank Thelen(Publisher)
...This enzyme allows DNA strands to be separated at a selected site, and new gene sequences to be inserted and then recombined. CRISPR-Cas is the most precise, inexpensive and straightforward tool for modifying the genetic code. The simplicity of CRISPR-Cas technology, in conjunction with the numerous options it opens up, has spawned a host of research projects and start-ups. The latter are working on ways to use CRISPR-Cas to achieve breakthroughs in the production of synthetic insulin, biofuels, proteins and antibiotics. The potentially great importance of CRISPR-Cas is well illustrated by the example of antibiotics, which, thanks to their ability to stave off the attack of pathogens in the human body, have saved millions of lives since their discovery about a century ago. But unfortunately, a growing number of pathogens are becoming resistant to antibiotics – which means that illnesses that are treatable today may become life-threatening at some point down the road. Several companies are using CRISPR-Cas methods to develop new types of antibiotics. For example, North Carolina-based Locus Biosciences has developed a technique, using a Cas3 enzyme, that targets specific pathogens and deactivates the pivotal elements of their DNA. Locus’s research's initial results were so promising that the pharma giant Johnson & Johnson made an $800 million deal with them. The role of DNA is to encode and store all genetic information of an organism. It contains the blueprints of all different enzymes that form a living being. You could compare DNA to a hard drive, or better a Read-only-Memory of biology. But reading the DNA and using the information to produce new cells is a multistep process that needs different types of RNA. In a process called transcription, a part of the DNA is read and copied into so-called Messenger RNA (mRNA), an inverted copy of this part of the DNA strain. This copy is transported to cell factories (ribosomes) located at the core (nucleus) of a cell...
eBook - ePubGenome Editing Tools and Gene Drives
A Brief Overview
- Reagan Mudziwapasi, Ringisai Chekera, Clophas Zibusiso Ncube, Irvonnie Shoko, Berlinda Ncube, Thandanani Moyo, Jeffrey Godfrey Chimbo, Jemethious Dube, Farai Faustos Mashiri, Moira Amanda Mubani, Duncan Maruta, Charity Chimbo, Mpumuzi Masuku, Ryman Shoko, Rutendo Patricia Nyamusamba, Fortune Ntengwa(Authors)
- 2021(Publication Date)
- CRC Press(Publisher)
...dCas9 is a catalytically dead variant developed as a sequence-specific transcriptional repressor and a genomic anchor. It is used for site-specific transcriptional regulation. This occurs when it is coupled with different kinds of genomic effectors. It also aides more precise DNA cleavage when it is coupled with other nucleases (Zhou and Deiters, 2016). This process is illustrated in Figure 5.5. Figure 5.6 A gRNA-directed double-strand DNA cleavage by Cas9 nuclease. PAM: Protospacer adjacent motif, a Cas9 recognition motif (5'-NGG-3') is required at the 3' end of the target DNA sequence; HNH and RuvC: Nuclease domains. Gene mutation by nonhomologous end joining (NHEJ) and gene insertion by homology-directed repair (HDR). (From Agrotis and Ketteler, 2015.) Applications of CRISPR CRISPR technology can be used to target prokaryotic cells and eukaryotic cells. Thus CRISPR can be utilized for gene editing in multiple organisms such as bacteria, yeast, plants, animals, and human cell lines (Lemay et al., 2017; Malnoy et al., 2016; Woo et al., 2015; Cho et al., 2013). CRISPR technology systems can be used to investigate gene function, gene development, and the development and the pathology of disease (Shao et al., 2016). The gene drive system can also be used to edit specific tissues such as the brain and liver tissues. CRISPR technology can be used to generate simultaneous multiple gene mutations (Li et al., 2013). CRISPR is gaining wide acceptance in CHO cell line engineering, synthetic biology, and genome engineering (Pennisi, 2013)...
eBook - ePubA Crack In Creation
Gene Editing and the Unthinkable Power to Control Evolution
- Jennifer A. Doudna, Samuel H. Sternberg(Authors)
- 2017(Publication Date)
- Mariner Books(Publisher)
...In effect, the CRISPR RNA molecule acted like a set of GPS coordinates, guiding Cas9 to a precise spot within the vast expanse of a long DNA molecule according to the matching letters in the CRISPR RNA and DNA. Here was a truly programmable nuclease, one that would be able to target any arbitrary DNA sequence using the same base-pairing rules—A goes with T, G goes with C, and so forth. For any twenty-letter sequence the guide RNA contained, Cas9 would find its matching counterpart in DNA and then cut. In the warfare waged between bacteria and viruses, Cas9’s function made perfect sense. Armed with a cache of RNA molecules derived from the CRISPR array, where snippets of phage DNA had been stored, Cas9 could readily be programmed to slice up corresponding sites within viral genomes. It was the perfect bacterial weapon: a virus-seeking missile that could strike quickly and with incredible precision. With Martin’s and Krzysztof’s results in hand, we were ready to tackle the next question: If bacteria could program Cas9 to cut up specific viral DNA sequences, could we, the researchers, program Cas9 to cut up other DNA sequences—viral or not—as we suspected? Martin and I were keenly aware of the developments in the gene-editing field and of the promise—but also the serious limitations—of the ZFN- and TALEN-based programmable nucleases. We realized, with no small sense of awe, that we had come upon a system that could be transformed into a far more straightforward gene-editing technology than anything previously discovered or developed. To turn this tiny molecular machine into a powerful gene-editing tool, we’d have to take one more step. So far, we had reduced a complex immune response into a simple set of moving parts that could be isolated, modified, and combined in different ways. What’s more, through careful biochemical experiments, we had deduced the molecular rules governing the functions of these different parts...
