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Current Application of CRISPR/Cas9 Gene-Editing Technique to Eradication of HIV/AIDS
Published in Yashwant Pathak, Gene Delivery, 2022
Prachi Pandey, Jayvadan Patel, Samarth Kumar
Cas9 is a nuclease of 1,368 amino acids, with two nuclease activity domains named HNH and RuvC. [59, 64–66] Each domain can cleave a DNA strand directed by a sgRNA complementary to the target DNA sequence (generally 20 nucleotides long). The prerequisite to be a target sequence is the presence of an NGG sequence (N: any nucleotides; G: guanine nucleotide, also called protospacer adjacent motifs (PAMs)) downstream (3′ end) of the target site. [59,64–66] In the nucleus, Cas9, sgRNA and target DNA form a complex. Then, HNH and RuvC domains each cleave a DNA strand [28–30]. The double strand DNA breaks are subsequently repaired by two different strategies. One is non-homologous end-joining (NHEJ) when there is no template, and the other is homology-directed repair (HDR) when there is a homologous nucleotide template present, such as ssDNA or dsDNA. (Figure 7.1).
Nucleic Acids as Therapeutic Targets and Agents
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
CRISPR-Cas gene editing is based on a complex of two crucial components, a single guide RNA fragment (sgRNA) which can be synthesized to contain the relevant sequence, and a protein (normally Cas-9, although many others are now used) that carries out the double-stranded cleavage. The complex scans DNA for the presence of a protospacer adjacent motif (PAM) which is 5′-NGG-3′ (N = any base) for Cas9, which originates from S. pyogenes. When a PAM sequence is detected, the complementary DNA strand is compared to the target-coded crRNA-derived guide region. If these sequences match, the DNA double strand is cleaved ~3 base pairs away from the PAM sequence by the Cas9 protein, thus introducing a double-stranded DNA break (DSB). Both cutting domains are located in the NUC lobe of Cas9, with the HNH domain cutting the strand complementary to the guide sequence (target strand), and the RuvC domain cutting the opposite strand (Figure 5.76).
Precision
Published in Lawrence S. Chan, William C. Tang, Engineering-Medicine, 2019
Double-stranded DNA Cutting. Guided by gRNA, the double-stranded DNA cleavage by Cas9 enzyme is conducted by two nuclease domains, namely the HNH domain and the RuvC-like domain. Whereas the HNH domain nuclease cleaves the DNA strand directly bound by gRNA, the RuvC-like domain nuclease cuts the opposite side of the double-stranded DNA.
A genomic sequence of the type II-A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system in Mycoplasma salivarium strain ATCC 29803
Published in Journal of Oral Microbiology, 2022
Harumi Mizuki, Yu Shimoyama, Taichi Ishikawa, Minoru Sasaki
The cas9 gene sequence of ATCC 29803 showed 98% identity to that of ATCC 23064. However, approximately 500 bp of sequence on the CRISPR array side showed no significant similarity to that of ATCC 23064. The Cas9 protein consists of a recognition lobe and a nuclease lobe, which includes several domains. The HNH and RuvC nuclease domains, which are contained in the nuclease lobe, are conserved, whereas the protospacer adjacent motif (PAM)-interacting (PI) domain is variable [31]. Approximately 500 bp of the cas9 gene sequence on the CRISPR array side in ATCC 29803, which was different from that of ATCC 23064, may include the PI domain encoding region. Therefore, when the cas9 gene of ATCC 23064 is not disrupted and coded, Cas9 functions as an endonuclease, and recognizable PAM sequences may differ between the two strains.
LsrB, the hub of ABC transporters involved in the membrane damage mechanisms of heavy ion irradiation in Escherichia coli
Published in International Journal of Radiation Biology, 2021
Xin Li, Lei Chen, Haitao Zhou, Shaobin Gu, Ying Wu, Bing Wang, Miaomiao Zhang, Nan Ding, Jiaju Sun, Xinyue Pang, Dong Lu
Based on KEGG annotation classifications, 74 genes of SHABCs were assigned to nine KEGG pathways (Table S8). Besides of 54 genes annotated in the ‘Membrane transport’ belonged to ‘Environment Information Processing (EIP)’, 11 genes (oppA/B/C/D/F, ydcS/T/U/V, ddpA and lsrB) were annotated in the ‘Quorum sensing (map02024)’ of ‘Cellular community – prokaryotes’ belonged to ‘Cellular Processes’. Three genes (gltL, gltK, and gltJ) were annotated in the ‘Two-component system (map02020)’ of ‘Signal transduction’ of EIP. Four genes (ruvB, ruvA, ruvC, and uvrA) were annotated in the ‘Replication and repair’ belonged to ‘Genetic Information Processing (GIP)’. Among the four genes, ruvA involved in the ‘Nucleotide excision repair (map03420)’, and the other three genes involved in the ‘Homologous recombination (map03440)’ of GIP. Moreover, five genes (oppA, oppB, oppC, oppD, and oppF) were gathered in ‘Drug resistance: antimicrobial’ of ‘Human Diseases (HD)’ (Fig. S2A). For the full information of genes annotation, see Table S8.
In vitro and in vivo efficacy of Caenorhabditis elegans recombinant antimicrobial protein against Gram-negative bacteria
Published in Biofouling, 2019
Dilawar Ahmad Mir, Krishnaswamy Balamurugan
The proteomic data analysis revealed that the ABF-1 protein affected cell cycle associated proteins (Tables 2 and 3) (RuvB helicase, RecA, mukF, endonuclease-VIII, Rho, rpoC, yajQ, cspA, greA, t1627, dnaB and rcsB). Among these, RuvB is a helicase protein that mediates DNA Holliday junction migration by localizing the denaturation and reannealing and RecA proteins play a role in homologous recombination, promoting synapsis, heteroduplex formation, and strand exchange between homologous DNAs (Iype et al. 1994). The mukF protein is involved in chromosome condensation, which is key step for cell division (Yamazoe et al. 1999). The endonuclease-VIII (NEI) protein acts as a DNA glycosylase that recognizes and removes DNA bases damaged by oxidation or mutagenic agents (Jiang et al. 1997). Collectively, these proteins play an important role in DNA damage, DNA recombination, DNA repair, SOS response and damaged DNA binding (Igarashi and Ishihama 1991; Borukhov et al. 1992; Bae et al. 2000; Skunca et al. 2013).