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Host Defense and Parasite Evasion
Published in Eric S. Loker, Bruce V. Hofkin, Parasitology, 2023
Eric S. Loker, Bruce V. Hofkin
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.
Differential Genetic Diagnosis between Leiomyoma and Leiomyosarcoma
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Alba Machado-Lopez, Aymara Mas
At the molecular level, myometrial tumors show unbalanced karyotypes as well as non-specific and complex alterations, such as SNVs, small indels, amplifications, and gene fusions (36,37). Specifically, up to 50% of LM have cytogenetic abnormalities, mainly affecting 6p21, 7q, and 12q15 chromosome regions as the main drivers, whereas point mutations in gene MED12 are detected in up to 70% of tumors (38). Chromosomal rearrangements between HMGA1/HMGA2 and RAD51B, or between COL4A5 and COL4A6 are also frequent in LM, mainly resulting in overexpression of these genes or reduced expression of CUX1 or CUL1 due to 7q deletions (25). Furthermore, heterozygous mutations in the FH (fumarate hydratase) gene can cause Reed's syndrome, also known as hereditary leiomyomatosis, and renal cell cancer syndrome, which is characterized by multiple cutaneous and uterine leiomyomas (Figure 25.3).
Advances in Genome Editing
Published in Yashwant Pathak, Gene Delivery, 2022
The type II CRISPR/Cas9 system, which relies on a single Cas protein from Streptococcus pyogenes (SpCas9) targeting specific DNA sequences and is hence an appealing gene editing tool, is the most commonly utilized subtype of CRISPR systems (Hsu et al., 2014; Mir et al., 2018; Tang and Fu, 2018; Wang et al., 2020). The CRISPR/Cas9 system is made up of two parts: a single-stranded guide RNA (sgRNA) and a Cas9 endonuclease. The sgRNA often provides a specific 20-base-pair (bp) sequence befitting the target DNA site in a sequential manner, and this must be accompanied by a short DNA sequence upstream integral for Cas9 protein suitability, known as the “protospacer adjacent motif” (PAM) of a “NGG” or “NAG” protein. Cas9 accurately cleaves the DNA to induce a DSB after the sgRNA attaches to the target sequence via Watson–Crick base pairing. DNA-DSB repair mechanisms begin genome repair after the DSB. Targeted genomic alterations, including the inclusion of tiny insertions and deletions (indels), can be generated with the CRISPR/Cas9 system via NHEJ or high-fidelity HDR pathways.
What’s next in cancer immunotherapy? - The promise and challenges of neoantigen vaccination
Published in OncoImmunology, 2022
Alec J. Redwood, Ian M. Dick, Jenette Creaney, Bruce W. S. Robinson
The bedrock of most clinical6,7,8 and preclinical studies5,17,18 are small somatic variations such as single nucleotide variants (SNVs) that lead to missense mutations, and small insertions or deletions (indels). SNVs and indels are the primary focus of most studies because they are readily detectable by whole-exome sequencing (WES). SNV induce immunogenic changes when they result in a mutation that changes an amino acid residue at an MHC anchor point, allowing an otherwise silent antigen to be presented, and/or when the amino acid substitution changes the topography of the MHC/peptide complex allowing novel T-cell recognition. Either mutation is capable of generating novel T-cell responses, however, there is no consensus on which if either of these events is the most immunogenic or protective when employed in a vaccine.
Neurodevelopmental and transcriptomic effects of CRISPR/Cas9-induced somatic orco mutation in honey bees
Published in Journal of Neurogenetics, 2021
Zhenqing Chen, Ian M. Traniello, Seema Rana, Amy C. Cash-Ahmed, Alison L. Sankey, Che Yang, Gene E. Robinson
Honey bee Orco is a seven-transmembrane domain protein. The Cas9 single guide RNA (sgRNA) was designed to target a site in the second exon, 235 bps downstream of the start codon. This cleavage site is in the codon of the 79th amino acid residue, within the second transmembrane domain (Figure 2(A,B)). This design was intended to maximize the potential knockout (KO) of Orco function. Frameshift mutations in this location cause extensive alteration in all downstream domains, and the indels of amino acid residues could potentially disrupt the structure of the transmembrane domain to affect normal function. The sgRNA was in vitro transcribed and mixed with purified Cas9 protein to form a ribonucleoprotein (RNP) complex, which enables the immediate action of Cas9 when injected.
Genetic analysis of primary renal cell carcinoma to determine treatment approaches
Published in Expert Review of Precision Medicine and Drug Development, 2021
Viktoria Stühler, Steffen Rausch, Arnulf Stenzl, Jens Bedke
Advances in modern genomic techniques, especially in next-generation sequencing, have highlighted the diverse spectrum of both genetic and epigenetic alterations in RCC. The total tumor mutation burden (TMB) in RCC is on average only moderately high and often unrelated to ICI response in mRCC. These tumors show a high rate of clonal insertion or deletion mutations (indels), which are known to increase tumor neoantigen frequency and CD8 + T-cell activation. Nevertheless, there is still no evidence of an association between indels and an anti-PD-1 response in mRCC [62]. However, in IMmotion150 the markers of a potential immunogenicity like TMB, tumor neoantigen burden (TNB), tumor indel burden (TIB), and frameshift mutation burden (FMB) were not associated with an intratumoral T-effector gene signature or a clinical benefit in any of the three treatment arms [36]. In CheckMate 214 TMB (higher or lower than median), TIB (higher or lower than median), and HLA zygosity were not associated with OS in patients treated with ipilimumab plus nivolumab. However, a high tumor indel burden was associated with improved PFS in patients treated with sunitinib [25].