Bioengineering and the Idea of Precision Medicine
Emmanuel A. Kornyo in A Guide to Bioethics, 2017
The first and perhaps most popular system is known as CRISPR/Cas system. CRISPR is a bacterial adaptive system. Through the process of evolution, bacteria have developed this defensive mechanism against phage infections. When a virus infects bacteria, the CRISPR system incorporates foreign genomic materials and this becomes inheritable. Thus, the bacteria is able to develop immunity in future infections by recognizing the specific sites of the new infections and eliminate them at these recognition sites. The Cas9, in particular, can be used to cut genes at any loci within the genome or alter specific genes responsible for a particular pathway or a gene of interests. Currently, because of its versatility, the CRISPR Cas9 system has become one of the most popular molecular tools in bioengineering and specifically, gene modifications.10
CRISPER Gene Therapy Recent Trends and Clinical Applications
Yashwant Pathak in Gene Delivery, 2022
Clustered regularly-interspaced short palindromic repeats (CRISPR) is a recent and powerful genetic editing tool. In the past decade, CRISPR–Cas9 technology is widely acknowledged for playing a substantial role in the field of biotechnology for editing the genome of any organism in the treatment of a variety of complex systemic diseases and for many other purposes. ‘CRISPR–Cas’ acronym stands for clustered regularly interspaced short palindromic repeats–CRISPR-associated genes. It is interesting to know that CRISPR sequences are an important component of the immune systems of simple life forms like bacteria and other microbes. The immune system is in charge of protecting the health and wellbeing of an organism. This genetic organization prevails in prokaryotic organisms and assists in the development of adaptive immunity because of a protein called Cas9 nuclease, which splits a specific target nucleic acid sequence of unknown invaders and destroys them. This mode of action has acquired the attention of researchers to understand the prospects of CRISPR–Cas9 technology. There are many future aspects and potential applications of CRISPR–Cas9 technology required mainly for the treatment of dreadful diseases, crop improvement, and genetic improvement in human beings. However, safety measures are implemented on this technology to avoid misuse or ethical issues [1].
Laboratory techniques to study the cellular and molecular processes of disorders
Louis-Philippe Boulet in Applied Respiratory Pathophysiology, 2017
Clustered regularly interspaced short palindromic repeats (CRISPR) refers to the natural occurrence of a DNA repeating sequence separated by a nonrepeating sequence, called spacer, in the prokaryotic genomes [50] (Figure 3.11). It is a defense mechanism used by prokaryotes to fight off viral infection [51,52]. Briefly, a spacer corresponding to a previously exposed viral sequence is kept between repeating DNA sequences so that it can be used to recognize the same virus in the future. To recognize and defend against a viral infection, the prokaryote transcribes the sequence into an RNA strand and together with a Cas enzyme, the complex drifts around inside the cell. Once a viral sequence matches the RNA sequence, the Cas enzyme snips the viral DNA and prevents the virus from replicating. Of the various Cas enzymes, Cas9 is the best known and it comes from Streptococcus pyogenes [53]. The derived gene-editing system is thus known as the CRISPR-Cas9 system. Briefly, this gene-editing method begins with a Cas9 enzyme guided to a specific DNA site with the help of a customized 17–20-nucleotide long guide RNA (gRNA). Cas9 snips the targeted DNA at the precise location generating a double-strand break (DSB) [54]. The cleaved DSB can be repaired by an error prone nonhomologous end joining (NHEJ) mechanism and generates random microinsertion or microdeletion (INDEL) mutations, or it can be replaced with a gene modification by homology-directed repair (HDR) in the presence of donor template [55]. Hence, NHEJ and HDR can be adapted to either silence a gene or replace a gene, respectively [56].
Layer-by-Layer technique as a versatile tool for gene delivery applications
Published in Expert Opinion on Drug Delivery, 2021
Dmitrii S. Linnik, Yana V. Tarakanchikova, Mikhail V. Zyuzin, Kirill V. Lepik, Joeri L. Aerts, Gleb Sukhorukov, Alexander S. Timin
Delivery of genome-editing (GE) tools via non-viral carriers has become a widely studied research topic. LbL technology can promote nucleic acid delivery methods that can be used for non-viral delivery of genome-editing tools. One of the most promising GE methods is CRISPR/Cas9, for which Jennifer Doudna and Emmanuelle Charpentier recently received the Nobel prize. The Cas9 nuclease is targeted to a specific site in DNA with the help of a guide RNA (gRNA) sequence. Cas9 then makes a double-strand break (DSB) at the intended site, which is followed by the activation of DSB repair systems. The induced break can be repaired by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), homology-mediated end joining (HMEJ), or homologous recombination (HR). Reparation of a double-strand break in targeted genes can result in deletions, insertions, or point mutations.
Cystic fibrosis – Ten promising therapeutic approaches in the current era of care
Published in Expert Opinion on Investigational Drugs, 2020
Ranjani Somayaji, Dave P Nichols, Scott C Bell
The best known gene-editing strategy is the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 whereby DNA is inputted using a specific technology to correct the mutated sequence of the CFTR gene [79]. The CRISPR/Cas9 system has been adapted from a host defense system that naturally occurs in bacteria whereby they obtain DNA fragments from invading viruses (i.e. bacteriophages) and create segments called CRISPR. These segments serve as a memory and can be used to target the virus upon re-infection to disable it. Similarly, in the lab, a small piece of RNA is created which can bind to a DNA sequence in the genome of human cells as well as to the Cas9 enzyme (or other enzymes in some cases) to cut at a specified location and edit accordingly. This approach has the potential advantages of life-long expression of the corrected gene in that cell or its progeny and reduced risk of insertional mutagenesis. The first studies using organoid-based methodology to explore CRISPR/Cas9 as a potential CF therapy were undertaken in 2013 and demonstrated that gene-editing led to corrected allele expression and a fully functioning organoid providing a proof of concept for this technology [80]. Subsequent studies of CRISPR/Cas9 as well as others including antisense-oligonucleotide-mediated and mRNA-mediated therapies have been conducted using various in vivo models and have demonstrated some success in relation to corrected allele expression and cell/organoid functioning [71,81,82,83].
CRISPR-based biosensing systems: a way to rapidly diagnose COVID-19
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Majid Vatankhah, Amir Azizi, Anahita Sanajouyan Langeroudi, Sajad Ataei Azimi, Imaneh Khorsand, Mohammad Amin Kerachian, Jamshid Motaei
CARP (Cas9/sgRNAs-associated reverse PCR) has been developed using the high specificity of Cas9 endonuclease and the high sensitivity of the PCR method [78]. CARP identifies target DNA in three stages. In the cleavage stage, the target DNA is cut simultaneously on two sites by Cas9 after identification of specific sequences with a pair of sgRNAs. In the ligation stage, the T4 DNA ligase ligates the cleaved DNA into intermolecular concatenated linear DNA or intramolecular circular DNA. The PCR stage is the proliferation of target DNA with a pair of reverse primers in traditional PCR or qPCR. Due to the reverse orientation of primers, target DNA amplification occurs after the cleavage and ligation stages. The CARP platform identified HPV16 and HPV18 viruses from other HPV subtypes [78]. ctPCR3.0 (CRISPR or Cas9-sgRNA typing PCR version 3) is a combination of Cas9, a pair of sgRNAs, a qPCR technique, and an isothermal incubation stage before qPCR that successfully detects target DNA in 2 h. ctPCR3.0 was able to detect the L1 and E6-E7 genes of HPV16 and HPV18 in clinical samples [79]. The LoD in the CARP and ctPCR3.0 platforms was 2 pg and 1.8 × 102 copies of target DNA, respectively [78,79]. These studies show that Cas9-based biosensing systems, as rapid, sensitive and cost-effective methods, have a high potential for virus detection.
Related Knowledge Centers
- Crispr
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