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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
Chimeric single guide RNA (sgRNA) is built by hybridization of the 5′ end of the tracrRNA with that of the 3′ end of the crRNA, subsequently forming a single guide RNA, which has a great potential in genome engineering as it can decompose any target DNA sequence. Type II is made up of two parts, Cas9 and sgRNA. The sgRNA guides the Cas9 endonuclease and identifies G-rich PAM (i.e., 5′-NGG) and then recognizes the target DNA sequence that are located in the upstream position of the PAM sequence and causes dissolving of the target DNA. As a result of this, the strands undergo directional separation, upstream to the PAM forming an R-loop. Later, the siRNA strand is incorporated and, thereby, the formation of the RNA–DNA heteroduplex takes place. The duplex is created by base pairing of the ~ 20nt spacer sequence of the sgRNA with that of the proto spacer of the target DNA because they are complementary to each other. One domain of the Cas9 enzyme, HNH, slices the DNA sequence that is complementary to the sequence (target sequence) present in guide RNA, while the other domain of the enzyme, RuvC, slices the sequence that is non-complementarily to the sequence (non-target sequence) present in guide RNA. This way, the two domains cleave the RNA–DNA hybrid at a spot three bp upstream to the PAM, and the consequence of cleavage is the formation of a double-strand break (DSB) with blunt ends [42–45, 47–51].
rDNA: Evolution Over a Billion Years
Published in S. K. Dutta, DNA Systematics, 2019
Mapping of the rDNA regions of T. aestivum was first carried out using a combination of R-loop analysis and hybridizing 125I labeled-rRNA species to digests of genomic DNA.81 These studies indicated a basic rDNA repeat unit of approximately 9.5 kb as defined by EcoRI, and that a Bam HI site in the large (26S) rRNA gene was not accessible to digestion in all repeating units. The heterogeneity within the wheat rDNA was more clearly defined by Gerlach and Bedbrook,93 who showed 3 length variants (9.0, 9.1, and 9.4 kb) existed; these authors also argued that methylation contributed to the lack of digestion of certain Bam HI sites in genomic DNA. Cloning of the 9.0 and 9.1 kb length variants was achieved by Gerlach and Bedbrook93 using DNA enriched for rDNA genes on actinomycin-D/CsCl gradients; EcoRI segments of DNA were inserted into the EcoRI site of pACY184. The detailed structure for one of these clones, pTA250, as derived by Appels and Dvorak94 is summarized in Figure 6. The spacer region was found to be dominated by the presence of 11 repeating units which are defined by the restriction enzymes Hae III and Hha I and are 131 to 133 bp in length. The repeating units are very similar as determined by sequencing 2 of the repeating units maximally different from each other by cross-hybridization criteria; they differed in 6 apparently randomly distributed positions. The 750 bp Hha I fragment near the start of the small (18S) rRNA gene very likely contains the site for transcription initiation by analogy with other systems, but this has not yet been demonstrated directly. We note that the sequence of 42 residues at the 3′-terminal of barley 18S rRNA has been determined,95 and in view of the extremely high conserved nature of this region (see Section V), this can be assumed to be the same as the respective region of wheat 18S, rRNA. The first 18 residues (3′OHGUACUAGGAAGGCGUCC) were determined directly from wheat 18S rRNA by Hagenbuchle et al.96
Targeting the DNA damage response in pediatric malignancies
Published in Expert Review of Anticancer Therapy, 2022
Jenna M Gedminas, Theodore W Laetsch
Ewing sarcoma is the second most common primary bone tumor of childhood and adolescence [80]. The tumor is defined by a chromosomal translocation fusing the EWSR1 gene to a member of the ETS family of transcription factors, most commonly FLI1. Outside of this translocation, the tumor has a very low mutational burden; however, the EWS-FLI1 transcription factor leads to the dysregulation of several genes involved in the response to DNA damage including CHK2, MAP4K2, ABL1, and ATM [81–83]. Ewing sarcoma is also known to have high levels of endogenous replication stress, making the cells highly reliant on the ATR pathway for the repair of the resultant DNA damage and maintenance of cell viability [29,84]. Gorthi et al. has shown that Ewing sarcoma cells have an increased accumulation of R-loops which are known to be associated with high levels of DNA damage [84]. This was associated with increased RNAPII binding at the same site as the R-loops, consistent with the knowledge that both EWSR1 and EWS-FLI1 regulate RNAPII elongation. Unresolved R-loops can lead to stalling of the replication fork, furthering the reliance on DNA damage repair [84].
An overview of potential novel mechanisms of action underlying Tumor Treating Fields-induced cancer cell death and their clinical implications
Published in International Journal of Radiation Biology, 2021
Narasimha Kumar Karanam, Michael D. Story
Interestingly, TTFields exposure in and of itself was shown to produce γ-H2AX foci, which is a marker of DNA damage as well as a marker for stalled replication forks, suggesting that TTFields not only delay DNA damage repair, but also induces replication stress. TTFields treatment downregulates the expression of MCM6 and MCM10 genes, essential components of the DNA replication complex and members of the FA pathway genes, leading to an elevated number of chromatid type aberrations. Furthermore, as part of the induction of replication stress, there is a decrease in the length of newly synthesized DNA and an increase in R-loop formation (Karanam et al. 2018, 2019). Mitosis and DNA damage pathways are tightly regulated through feedback mechanisms. By monitoring temporal gene expression changes associated with regulators of mitosis and DNA damage pathways, Karanam et al. showed that mitotic aberrations and DNA damage events while certainly linked to one another likely also occur independent of each other. These results established the role of TTFields in DNA damage repair and replication stress pathways. Key events in mitosis and DNA damage and replication stress pathways that are affected by TTFields are shown schematically in Figure 2.
Role of DNA damage and repair in radiation cancer therapy: a current update and a look to the future
Published in International Journal of Radiation Biology, 2020
Jingya Liu, Kun Bi, Run Yang, Hongxia Li, Zacharenia Nikitaki, Li Chang
An interesting aspect of DDR is the implication of RNA for the processing of DSBs. The notion of RNA-dependent DNA repair (RDDR) has been an upcoming potential strategy in cancer research and therapy, especially under the recent emergence of RNA therapies (Cao et al. 2019). The general idea as recently reviewed in (Bader et al. 2020), it is that RNA-interacting proteins play an important role in DNA Damage Response (DDR), and a number of different RNA-processing enzymes are normally recruited to the sites of chromatin with DNA damage and also reformed in response to different types of damage, supporting the scenario that these specific alterations can be used to modify accordingly repair outcome. RNA may mediates canonical DSB pathways, i.e. those that function in both normal and tumor cells. Moreover it is proposed an additional pathway, similar to single strand annealing HR sub-pathway, where instead of the D-loop, an R-loop is formed. R-loop refers to a three-stranding poly-nucleotide structure, consisting of a hybrid DNA:RNA and of a non-template single stranded DNA (Bader et al. 2020).