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Studying Brain Function Using Non-human Primate Models
Published in Tian-Le Xu, Long-Jun Wu, Nonclassical Ion Channels in the Nervous System, 2021
NHP genome editing techniques are still developing. Recently, precise exogenous gene knock-in NHPs via CPRISR/Cas9 128,129 has been tested in recent study (128,129). This technique can overcome the limitation of lentiviral vector-mediated gene transferring at random insertion and uncontrollable expression levels (130). More conditional expression lines, i.e., Cre lines, can be generated in the near future with the establishment of Tet-on system. These versatile NHP models demonstrate huge potential for providing researchers with further insight into the mechanisms of human development and human diseases125 (Figure 10.3).
Investigating the Role of Two-Pore Channel 2 (TPC2) in Zebrafish Neuromuscular Development
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sarah E. Webb, Jeffrey J. Kelu, Andrew L. Miller
Despite the fact that MOs have contributed greatly to the identification of hundreds of genes that are crucial for development, as well as to the elucidation of gene function, their use has recently been severely criticized due to their potential off-target effects (Eisen and Smith, 2008; Gerety and Wilkinson, 2011; Robu et al., 2007), as well as their failure to phenocopy the corresponding mutants (Kok et al., 2015). This led to the research community moving towards the use of permanent gene knock out techniques such as CRISPR/Cas9 gene-editing. However, reports now indicate that CRISPR/Cas9 might also be affected by off-targeting effects (Kuscu et al., 2014; Tsai and Joung, 2016). Indeed, it was recently suggested that the phenotypic discrepancies observed between some morphants and their corresponding mutants, might be due to the presence of genetic compensation in the mutants (Rossi et al., 2015). Thus, at least some of the differences observed between the morphants and the corresponding mutants might not just be attributable to the off-target effects of MOs; they might also be a result of the poor characterization of the mutants (Kok et al., 2015). However, with the appropriate controls, MOs can still be considered to be a reliable and valuable tool (Blum et al., 2015; Schulte-Merker and Stainier, 2014; Stainier et al., 2015, 2017). In addition, by using a combination of complementary transient-knock down (with MOs) and permanent-knock out (via CRISPR/Cas9), it is possible to obtain comprehensive data for a more thorough, comparable analysis.
The science of biotechnology
Published in Ronald P. Evens, Biotechnology, 2020
CRISPER/Cas9 is a clustered regulatory interspaced short palindromic repeat DNA sequence along with a CRISPER-associated protein enzyme with nuclease function. The Cas9 in CRISPER is formed from two short RNA molecules: a guidance RNA (gRNA) with a transactivating (tracrRNA) complex, which cleaves the DNA. Furthermore, another molecule involved in order for CRISPER to work is the protospacer adjacent motif (PAM), which needs to be adjacent to the acquired spacer sequence. Then, a desired DNA sequence can be inserted. In comparing gene editing methods, a challenging list of characteristics is desirable as follows: simplicity in design, engineering feasibility, multiple genome editing, specificity in targeting, efficiency in operations, reasonable cost, minimal off-target effects, minimal immune reactions, and no cytotoxicity. The uses are quite manifold: diagnostic utility, clinical usage, gene therapy, possible epigenetic utility, possible gene knock-out activity, RNA editing possibility, and mitochondrial DNA activity. The science and applications of CRIRPER/Cas9 gene editing is advancing rapidly creating new exciting treatment modalities with broad applications.
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/Cas9-mediated knock-in genetic editing of immune cells is complex. Because of the low transfection efficiency of these cells, according to Lichen Zhang and colleagues. Virus vectors are widely used to boost cells expressing the CRISPR/Cas9 technology in immune cells, which typically results in undesired cell interference. As CRISPR/Cas9 was coupled after electroporation, the previously generated dual fluorescent reporters spontaneously formed [120]. This sort of gene knock-in cell may be used in several molecular studies. For exchange, a lot of experimental analyses have employed reporter genes or intermediates. CRISPR/Cas9-based gene exchange is a faster and more efficient strategy for comparing viral gene expression than other classic recombination technologies [121].
Mechanism of Action of Mesenchymal Stem Cells (MSCs): impact of delivery method
Published in Expert Opinion on Biological Therapy, 2022
Luiza L. Bagno, Alessandro G. Salerno, Wayne Balkan, Joshua M. Hare
As described above, CRISPR/Cas9-mediated gene knockdown in MSCs has proved effective in treating diseases such as myocardial infarction [102]. The converse, targeted gene knock-in, where a gene is inserted into the genome via homologous recombination, resulting in overexpression of the protein, can also be beneficial. Tilokee et al. demonstrated that paracrine engineering of human cardiac stem cells to overexpress SDF-1α enhances recruitment of endogenous stem cells, promotes myocyte/vessel formation, and salvages reversibly damaged myocardium to enhance cardiac repair in a mouse model of MI [103]. These and other cell pre-conditioning and genetic modifications are promising options for augmenting MSC- and other stem cell-based therapies [104] and represent viable approaches for improving treatment for a wide variety of diseases.
SpCas9-expression by tumor cells can cause T cell-dependent tumor rejection in immunocompetent mice
Published in OncoImmunology, 2019
Reham Ajina, Danielle Zamalin, Annie Zuo, Maha Moussa, Marta Catalfamo, Sandra A. Jablonski, Louis M. Weiner
These exciting advantages over prior gene editing techniques have fostered the concept of employing CRISPR-Cas9-mediated genome editing in the research and development of therapeutics. The system has already been successfully employed for in vitro gene knock-in and knock-out studies.9,10 Also, it has been utilized to investigate transcriptional regulation.2 CRISPR-Cas-induced embryo modification has recently led to the development of precisely engineered mice.11 Such animal models represent important additions to the research on the impact of certain genes on disease onset and progression. Furthermore, by harnessing the ability to change a faulty gene itself, the introduction of CRISPR-Cas9 technology could be employed as a therapeutic for hereditary or mutation-based conditions.12