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Health Professionals and Modern Human Research Ethics
Published in Howard Winet, Ethics for Bioengineering Scientists, 2021
Any gene-based disease is a potential target, and there are more than 50 companies pursuing applications that would eliminate unwanted genes, add wanted genes, reduce the activity of overactive genes or increase the activity of underactive genes. Unwanted side effects include off-target effects due to guide RNA matching with untargeted domains and promiscuous action of Cas9 that cleaves untargeted domains. As a potential eugenics tool, this technology presents ethical challenges that could complicate its regulation. It is not an office-based treatment. But if it becomes one, regulation will be difficult.
Nucleic Acids as Therapeutic Targets and Agents
Published in David E. Thurston, Ilona Pysz, 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).
Genetic Limitations to Athletic Performance
Published in Peter M. Tiidus, Rebecca E. K. MacPherson, Paul J. LeBlanc, Andrea R. Josse, The Routledge Handbook on Biochemistry of Exercise, 2020
A significant recent advancement in the field of gene therapy is the discovery and development of the CRISPR–Cas9 system for modifying DNA. The Cas9 protein assembles with a guide RNA, enabling DNA binding and cutting much more precisely than has previously been possible in humans. This opens up possibilities for gene therapy but also for gene doping. The CRISPR–Cas9 system can create either permanent or temporary changes to the genome causing insertion, deletion, or replacement of gene(s), single-base changes, or gene suppression or activation (36). New, potentially even better, genome editing tools are also emerging, such as prime editing, CRISPR–Cas3, and EvolvR (36). Whilst gene therapy is not currently mainstream, we move ever closer to that scenario.
Challenges and opportunities when transitioning from in vivo gene replacement to in vivo CRISPR/Cas9 therapies – a spotlight on hemophilia
Published in Expert Opinion on Biological Therapy, 2022
Oscar G Segurado, Ruhong Jiang, Steven W Pipe
The sensitivity and specificity of CRISPR/Cas9 are dependent on the gRNA sequence specificity, as such much effort has been put into improving the gRNA currently being used in gene-editing therapies. The use of modified guide RNAs, like truncated gRNA, which has been shortened using a crRNA-derived sequence or gRNA that has been lengthened by two guanine (G) nucleotides, has seen increasing use in attempts to combat off-target effects [30–32]. The use of paired nickases, which combines a gRNA with paired Cas9 nickases, has been shown to greatly reduce off-target effects [17,23,31]. For example, Cas9 nickase, which is a mutated form of SpCas9, is responsible for creating a ‘nick’ in a single strand and thereby requiring two nickases to properly break both strands of DNA [23,32]. Several new Cas9 variants have been designed recently with hopes to improve on-target activity, reduce off-target effects, and create permanent gene modification. Engineered high-fidelity SpCas9 has been developed, followed by hyper-accurate Cas9 when it became clear that high-fidelity SpCas9 had poor activity at certain loci [33,34].
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.
The CRISPR revolution and its potential impact on global health security
Published in Pathogens and Global Health, 2021
Kyle E. Watters, Jesse Kirkpatrick, Megan J. Palmer, Gregory D. Koblentz
Second, another challenge with genome-editing technology for treatments of biothreat agents is the high propensity of those agents to mutate. Indeed, directly targeting a pathogen creates the possibility of stimulating its intrinsic mutation rate [110]. As previously stated, many of the agents identified by the CDC as biosecurity threats are RNA viruses, which as a group tend to rapidly mutate, making vaccine and traditional drug development difficult. Targeting specific strains, or incomplete clearing of virus from the host, could result in an artificial selection for viruses with mutations or sequences that avoid CRISPR targeting. The same would be true for other pathogens as well. The mutation of bases that are critical for CRISPR targeting, such as those in the seed region of the target sequence, or bases in the protospacer adjacent motif [111] could potentially lead to inactive treatments. Care must be taken to choose guide RNAs that target the most conserved regions of a gene to avoid mutation issues, while working within the restrictions placed by the PAM sequence required by each effector. Similarly, the appearance of single nucleotide polymorphisms within the human population presents a challenge when designing prophylactic genome-editing treatments, as certain guide sequences many only result in proper targeting in a subset of the global population.