دانلود کتاب ویرایش ژن با استفاده از CRISPR-Cas

The completion of the human genome sequencing represents a significant milestone in the realm of genetic research focused on diseases. Nevertheless, there exists a notable gap in our understanding of the roles of genes and their variants, primarily as we currently lack the know-hows necessary for precise gene sequence modifications, which are vital for experimentally assessing their molecular functions. The term genome editing denotes the targeted alteration of genomic DNA across a range of cell types and organisms. This technique includes the substitution, insertion, and deletion of DNA, which can result in the silencing of target genes, acquisition of new genetic traits, and the correction of deleterious gene mutations (Charpentier and Marraffini, 2014). In recent years, a remarkable progress in life sciences has been witnessed, establishing genome editing as the primary method for exploring gene function, shedding light on the pathogenesis of genetic disorders, identifying innovative targets for gene therapy, and advancing agricultural crop varieties (Cox et al., 2015). Molecular scissors that have been engineered for specific genomic modifications hold substantial potential for conducting functional analysis. These advancements could significantly enhance our understanding about the molecular basis of diseases and lead to the development of novel therapeutic approaches. A variety of platforms for these molecular scissors have been created, including zinc finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), and the recently introduced clustered regularly interspaced short palindromic repeats (CRISPR), along with CRISPR-associated protein 9 (Cas9). Earlier, scientists had discovered the CRISPR-Cas protein system as an innovative microbial defense system against bacteria and archaea (Bolotin et al., 2005). In bacteria, it protects from bacteriophages (bacterial viruses) and mobile genetic elements (transposons); hence, it works as an adaptive immune system in various prokaryotes (Mojica et al., 2009; Jinek et al., 2012). Additionally, in bacteria it was found that after phage infection, a spacer sequence was present in the genome, and alteration in these sequences affects phage growth within the target cell (Barrangou et al., 2007). The CRISPR-Cas protein system is a ribonucleic acid (RNA)–mediated selective deoxyribonucleic acid (DNA) editing or degradation system; that is, it is an RNA-mediated deoxyribonuclease (DNase). The CRISPR system consists of two foremost parts: CRISPR and Cas. The CRISPR is a shortsized guide RNA (gRNA), approximately 20–30 nucleotides, and is complementary to the target DNA, whereas Cas is an endo-DNase protein. The gRNA, also known as CRISPR-RNA (crRNA), guides the Cas protein to degrade or edit the target nucleic acid (DNA). In CRISPR-Cas systems, there are two main parts, the crRNA and the Cas, working interdependently in a target-specific manner. In this system, the Cas protein works like a molecular scissor, cutting/breaking the target DNA at specific locations that exactly matches the sequence of the crRNA. Hence, in CRISPR-Cas systems, the target DNA sequence is the focus of the crRNA; therefore, it is designed by utilizing bioinformatic techniques to decrease the nonspecificity. After the double-strand DNA break is triggered by Cas, multiple changes can be introduced at the site with the help of cellular repair processes using either homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways (Fu et al., 2021). In the beginning, the genome editing functions of CRISPR-Cas systems were unknown because of the vast defense systems in bacteria and archaea (Bolotin et al., 2005; Mojica et al., 2009; Jinek et al., 2012). Hence, earlier research scientists were focused on the reason behind the bacterial defense system. However, after the discovery of the CRISPR-Cas system, the genome editing process has witnessed a revolution in the molecular science field, and it is now becoming a leading technique garnering significant interest (Rao et al., 2022). A revolutionary age in genome (DNA) editing has been ushered in by the CRISPR-Cas system because it is fast, cheap, and has extraordinary precision and versatility. It is a major breakthrough in the field of molecular science because it performs better in terms of cost-efficiency and is relatively easier to use than previous genome editing methods, like ZFNs and TALENs. Further, it also demonstrates that this system improves editing outcomes due to its high efficacy and less off-target effects. Thus, due to these benefits, CRISPR-Cas has been widely used and has a more significant impact when compared to previous ZFNs and TALENs (Gaj et al., 2013). Thus, the changes that are introduced at the target site by the CRISPR-Cas system are incredibly useful for basic research, medicine, and crop improvement because of its precise and programmable genome editing capabilities (Li et al., 2023). Hence, it is used in diverse fields, including drug development science, human gene therapy, medical research, plant science, and in agricultural development (Liu et al., 2021)…