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CRISPER Gene Therapy Recent Trends and Clinical Applications
Published in Yashwant Pathak, Gene Delivery, 2022
Prachi Pandey, Jayvadan Patel, Samarth Kumar
The purpose of carrying out the CRISPR research is not to cut the DNA strand, but CRISPR is used basically to initiate the DSB to those DNA strands to change the genetic sequence of the nucleotides on that DNA strand. CRISPR will not be useful for such a purpose of changing the specific nucleotide because the only function it can perform is cutting the DNA strand. Thus, along with this, a procedure of fixing and editing the fragmented DNA strands is essential for the DNA to perform its function again and initiate changes to the targeted DNA sequences. There are two distinct mechanisms to edit and fix the broken DNA sequence, Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) [81, 82].
Nucleic Acids as Therapeutic Targets and Agents
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
An alternative strategy is to introduce a homologous DNA “donor sequence” into the cell at the time of the double-strand break so that it can be incorporated into the homology-directed repair pathway and added to the genomic sequence. Using this approach, a new DNA sequence (or even a whole gene) may be inserted into a cell’s genome, which can be used for functional genomics experiments, target validation, the creation of knock-in cell lines with fusion tags, promoters or reporters integrated into endogenous genes, and the creation of cell lines that produce higher yields of proteins or antibodies. For example, Sangamo Therapeutics Inc developed SB-728-T, a ZFN-based agent that can modify and knock-out the gene encoding CCR5, the major cell-surface co-receptor used by HIV to infect cells of the immune system. In clinical trials, CD4 T cells are extracted from individuals with HIV, edited at the CCR5 gene using the Zinc Finger Nuclease technology, and then expanded and re-infused into the patients. The company was hoping that this could achieve control of HIV viral load, but so far it has not been possible to modify sufficient numbers of CD4 T cells to produce a clear clinical benefit. Therefore, in 2019 it was announced that commercial development of SB-728-T would cease but with some investigator-initiated studies continuing.
Molecular Biology Tools to Boost the Production of Natural Products
Published in Luzia Valentina Modolo, Mary Ann Foglio, Brazilian Medicinal Plants, 2019
Luzia Valentina Modolo, Samuel Chaves-Silva, Thamara Ferreira da Silva, Cristiane Jovelina da-Silva
In CRISPR/Cas9 technology, the cleavage of double-strand DNA is assisted by a single nuclease (Cas9) that is guided to the target by a small RNA through Watson–Crick base pairing (Gasiunas et al., 2012). This method is of relatively low cost, versatile, easy to perform, highly specific and efficient that can be used for both forward and reverse genetics. This gene editing system enables the generation of an organism with a “clean” modified genome, which would essentially make it a nongenetically modified organism (Abbai et al., 2017). CRISPR-Cas9 technique was developed from the observation of the defense system, naturally occurring in bacteria. One of the defense mechanisms exhibited by prokaryotes is the regularly interspaced short palindromic repeats (CRISPR). The locus received this name because it is constituted of sequence repeats of 29 base pairs separated by variable sequences of 32 nucleotides referred to as spaces (Ishino et al., 1987). The sequencing of some bacteria and virus genomes shed light on the compatibility of the CRISPR spaces with phages and plasmids. Currently, it is known that CRISPR is involved in a protection system highly conserved among prokaryotes: 45% of bacteria and 90% of Archaea possess CRISPR locus (Nemudryi et al., 2014). For instance, bacteria capture DNA fragments from an invading virus to generate small DNA sequences called CRISPR arrays that will function as a “memory” of the pathogen attack for future self-defense, in case the virus and related pathogens try to infect the bacteria again. The bacterial enzyme Cas9, breaks down the viral DNA to prevent its action (Barrangou et al., 2007). The system CRISPR-Cas9, for the sake of genome editing, works in a similar way. First, it is synthesized as a short RNA sequence (about 20 base pair long) containing a guiding sequence that is complementary to a specific sequence of the DNA to be edited. A complex formed between Cas9 and the short RNA sequence then “search” for the complementary region in the target DNA and intercalates the DNA double strand at that point to indicate the region where Cas9 is required to break down (Hsu et al., 2014). The host cell repairing machinery can take care of the damaged DNA, adding or suppressing DNA fragments or even substituting DNA fragments for customized DNA. The DNA repair may occur by two mechanisms: (1) nonhomologous end-joining or (2) homology-directed repair (Zych et al., 2018). In the first mechanism, the DNA ends become adjacent to recombine without a template, which in turn can lead to insertions/deletions, altering the gene open reading frame. As for the homology-directed repair strategy, disrupted sequences are resynthesized, using as a template a homologous sequence throughout the genome (Wyman and Kanaar, 2006). Thus, homology-directed repair is useful to promote site-directed genome editions, while nonhomologous end-joining is used to rearrange chromosomes or generate functional knockouts (Montano et al., 2018; Tan et al., 2018).
SOD1-targeting therapies for neurodegenerative diseases: a review of current findings and future potential
Published in Expert Opinion on Orphan Drugs, 2020
John P. Franklin, Mimoun Azzouz, Pamela J. Shaw
First described in 2013, the CRISPR/Cas9 gene editing system is derived from bacterial cell defense mechanisms [102,103]. Briefly, it comprises two components: a guide RNA (gRNA), complementary to the region of genomic DNA to be targeted, and a Cas9 nuclease which is directed by the gRNA to form double strand breaks at a precise location. The regions which can be targeted with this system are dictated by the presence of specific nucleotide sequences called protospacer adjacent motifs (PAMs), which occur frequently throughout the genome. After a double strand break, endogenous cell machinery may repair the break using either non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ frequently leads to indel mutations, causing a frameshift and resultant gene knockout [104]. The homology-directed repair mechanism may also be exploited to correct a specific mutation when a template DNA strand is introduced to the cell at the same time. CRISPR/Cas9 has been heralded as a huge breakthrough for future gene therapy: it is hoped that monogenic disease may in future be cured or prevented through correction of mutations in somatic or germline cells respectively.
Ras-Mediated Activation of NF-κB and DNA Damage Response in Carcinogenesis
Published in Cancer Investigation, 2020
Few other honorable mentions of the vital DNA damage and repair pathways include Bloom syndrome protein (BLM), that in humans is regulated by BLM gene and possess both DNA-stimulated ATPase and ATP-dependent DNA helicase activities. Mutations may delete or modify the helicase motifs and may incapacitate its helicase function and is somatically altered which becomes the hallmark of a number of cancer types including colorectal cancer (195–198). Similarly Checkpoint kinase 2 (CHEK2) is another tumor suppressor gene that gives the protein CHK2 and regulates genome instability. It is needed in homology directed repair by regulating cell cycle checkpoints in such a way that DNA double strand breaks get correctly repaired. The erroneous activity of CHEK2 is linked with cell cycle checkpoint errors and incorrect DNA repair and tumor development (199–201) (Figure 8).
An overview: CRISPR/Cas-based gene editing for viral vaccine development
Published in Expert Review of Vaccines, 2022
Santosh Bhujbal, Rushikesh Bhujbal, Prabhanjan Giram
AdV is a non-enveloped virus with a double-strand DNA genome virus including an icosahedral nucleocapsid that can not only infect both dividing but also non-dividing cells [152]. Due to their moderate packaging potential, adenoviral vectors (AdVs) can probably possess all components for genetic engineering, expressing both the Cas enzyme and one or more sgRNAs out of a single vector [153]. Large donor genetic sequences can also be co-deliveries to facilitate homology-directed repair. The additional benefit is that sgRNA and Cas enzyme are frequently expressed along with the same cell at a constant proportion, whereas Cas expression in the cell cycle is transitory because AdVs are non-integrating [154,155].