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
Host Defense and Parasite Evasion
Eric S. Loker, Bruce V. Hofkin in Parasitology, 2023
The CRISPR system, in addition to being a fascinating and effective means of protection of bacteria from their pathogens, is also worthy of mention for its exploitation by biologists as a powerful and specific gene-editing tool, including for host–parasite studies. The 2020 Nobel prize in chemistry was awarded to Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier for their ground-breaking discoveries with CRISPR gene editing. Applications of CRISPR technology relevant to parasitology are mentioned elsewhere in the book (e.g. Chapters 8 p. 411, and 9 p. 436). Basically, we have learned to make various DNA constructs bearing sequences encoding Cas enzymes and to introduce them into cells of many kinds, including human cells. Also included in the constructs are sequences that specifically match those of target genes we wish to modify or disable. Depending on the specific approach taken, the target gene can be disabled by addition or deletion of incorrect bases (an indel mutation), or modified to have a different function or altered expression (Figure 4.3). Here it must be stressed that this powerful technology has the potential to have “off-target” effects and alter genes other than those intended. Also, genomes of targeted cells may devise ways to protect themselves from such modifications. Many more sophisticated variations on this common theme are constantly being developed by the bioengineering community to effect precise and efficient gene-editing changes.
Nucleic Acids as Therapeutic Targets and Agents
David E. Thurston, Ilona Pysz in Chemistry and Pharmacology of Anticancer Drugs, 2021
CRISPR-Cas gene editing is based on a complex of two crucial components, a single guide RNA fragment (sgRNA) which can be synthesized to contain the relevant sequence, and a protein (normally Cas-9, although many others are now used) that carries out the double-stranded cleavage. The complex scans DNA for the presence of a protospacer adjacent motif (PAM) which is 5′-NGG-3′ (N = any base) for Cas9, which originates from S. pyogenes. When a PAM sequence is detected, the complementary DNA strand is compared to the target-coded crRNA-derived guide region. If these sequences match, the DNA double strand is cleaved ~3 base pairs away from the PAM sequence by the Cas9 protein, thus introducing a double-stranded DNA break (DSB). Both cutting domains are located in the NUC lobe of Cas9, with the HNH domain cutting the strand complementary to the guide sequence (target strand), and the RuvC domain cutting the opposite strand (Figure 5.76).
Novel technologies to characterize and engineer the microbiome in inflammatory bowel disease
Published in Gut Microbes, 2022
Alba Boix-Amorós, Hilary Monaco, Elisa Sambataro, Jose C. Clemente
Classic tools to genetically engineer bacteria use recombinant plasmids as cloning vectors to deliver genes of interest. Cloning vectors allow the production of large amounts of protein, but embody certain limitations including low efficiency of transformation and limitations of insert size. Recent discoveries and development of new tools, such as the CRISPR-Cas systems, have revolutionized the genetic engineering scene, offering an enormous potential to engineer genomes with greater efficacy than previously achieved.223 CRISPR (clustered regularly interspaced short palindromic repeats) and its associated Cas proteins are tools derived from the prokaryotic immune system that have been co-opted as genetic editing tools.223,224 This methodology has been applied to bacteria and yeasts to modify their functional repertoire, exploited for industrial applications or used for the direct removal of specific genes or pathogens.222–225 Among the different CRISPR systems, CRISPR-Cas9 and CRISPR-Cas12a (also known as Cpf1) are two major nucleases that have been used in bacterial genetic editing experiments. By inserting a guide RNA sequence targeting a specific region of the bacterial DNA, Cas nucleases introduce a break in the pathogen’s genome which allows the removal of specific genes or causes bacterial death.223,226,227 If a template DNA is provided, the genomic break introduced by the Cas nucleases can be repaired by homologous recombination, inserting the new DNA fragment into the bacterial genome.
An overview: CRISPR/Cas-based gene editing for viral vaccine development
Published in Expert Review of Vaccines, 2022
Santosh Bhujbal, Rushikesh Bhujbal, Prabhanjan Giram
CRISPR/Cas gene editing requires two basic components: a Cas enzyme and guide RNA. These components are associated to form a ribonucleoprotein (RNP) complex. CRISPR/Cas system involves different types of Cas enzymes, among them, Cas9 and Cas12a are the most widely used for gene editing. The Cas9 enzyme is a nonspecific type II CRISPR locus, derived spCas9 from Streptococcus pyogenes SF370 [14]. The Cas12a enzyme, also known as Cpf1, is derived from Acidaminococcus sp. (AsCas12a) and Lachnospiraceae bacterium (LbCas12a) [18]. The RNP Complex: Cas protein and gRNA together shown to have a bacterial defense system and also some antiviral effect [19]; this is followed by three steps: 1) Acquisition- in which newly infected viral DNA invades the leading CRISPR locus, 2) Expression- in which the Cas gene is expressed and the CRISPR system is transcribed into a precursor CRISPR-RNA (Pre-crRNA) [20], which subsequently matures into a short mature crRNA with spacers and repeats. 3) Interference- if the virus DNA infects the bacteria again, the CRISPR/Cas9 system interferes, allowing the bacteria to keep a record of the infection and the CRISPR locus to serve as a genetic vaccination card for those bacteria [21,22].
CRISPR gene editing – what are the possibilities for respiratory medicine?
Published in Expert Review of Respiratory Medicine, 2022
A second way to avoid double-stranded breaks and potentially treat disease is epigenetic editing. This involves the use of CRISPR to target transcriptional regulators in precise regions of the genome in order to modify gene expression. An interrogation of 18 potential regulatory regions of the CFTR gene with 133 different gRNAs and the dCas9p300 transcriptional activator identified a single guide, gRNA40, which gave a moderate increase in the levels of CFTR mRNA in wild-type cells [9]. When tested on cells homozygous for the most common CF-causing mutation, F508del, the dCas9p300/gRNA40 resulted in a much larger increase in F508del CFTR mRNA. Whilst this increase in mRNA alone had no significant increase in the F508del CFTR ion channel activity, a synergistic interaction was observed with the CF modulator drug, VX809, almost doubling the short circuit current activity of the F508del CFTR ion channel relative to VX809 treatment alone. It will be interesting to see if dCas9p300/gRNA40 can upregulate mRNA expression of the other CF variants, particularly stop codon (PTC) variants where the CFTR mRNA is destabilized, and see if this leads to synergistic interactions with PTC read-through drugs in development. However, a potenital challenge in developing this approach for therapeutic application is that either long-term expression, or repeat dosing, of the dCas9p300 and gRNA would be required.