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CRISPR-Cas in Genomic Manipulation and Antimicrobial Resistance

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Microbiology".

Deadline for manuscript submissions: closed (31 January 2023) | Viewed by 6966

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Dr. Aixin Yan

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School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong
Interests: CRISPR-cas systems; bacteria; antimicrobial resistance

Special Issue Information

Dear Colleagues,

CRISPR-Cas-mediated genome editing has revolutionized the genetics and biomedical research in many model eukaryotic organisms on account of its efficiency, affordability, and accessibility. Although their applications to edit prokaryotes have been relatively unexplored and underrepresented due to the requirement of species-specific optimization for plasmid delivery and Cas proteins expression, a growing number of the CRISPR-Cas-mediated genome editing are established in model bacterial strains and species. Remarkably, since more than half of bacterial genomes harbor native CRISPR-Cas systems, various native type I CRISPR-Cas-mediated genome editing techniques are being established in both model and wildly isolated bacterial strains including clinical isolates and isolates of the human microbiome. Furthermore, the newly emerged compact systems, such as Cas12e, Cas12f, and Cas12j, are increasingly applied to edit bacterial genomes with efficiency and simplicity. This Special Issue is expected to share these developments and promote their applications to tackle the ever-growing problem of antimicrobial resistance.

Dr. Aixin Yan
Guest Editor

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Research

15 pages, 1964 KiB

Open AccessArticle

Prenyl Transferases Regulate Secretory Protein Sorting and Parasite Morphology in Toxoplasma gondii

by Qiang-Qiang Wang, Kai He, Muhammad-Tahir Aleem and Shaojun Long

Int. J. Mol. Sci. 2023, 24(8), 7172; https://doi.org/10.3390/ijms24087172 - 12 Apr 2023

Cited by 2 | Viewed by 1557

Abstract

Protein prenylation is an important protein modification that is responsible for diverse physiological activities in eukaryotic cells. This modification is generally catalyzed by three types of prenyl transferases, which include farnesyl transferase (FT), geranylgeranyl transferase (GGT-1) and Rab geranylgeranyl transferase (GGT-2). Studies in malaria parasites showed that these parasites contain prenylated proteins, which are proposed to play multiple functions in parasites. However, the prenyl transferases have not been functionally characterized in parasites of subphylum Apicomplexa. Here, we functionally dissected functions of three of the prenyl transferases in the Apicomplexa model organism Toxoplasma gondii (T. gondii) using a plant auxin-inducible degron system. The homologous genes of the beta subunit of FT, GGT-1 and GGT-2 were endogenously tagged with AID at the C-terminus in the TIR1 parental line using a CRISPR-Cas9 approach. Upon depletion of these prenyl transferases, GGT-1 and GGT-2 had a strong defect on parasite replication. Fluorescent assay using diverse protein markers showed that the protein markers ROP5 and GRA7 were diffused in the parasites depleted with GGT-1 and GGT-2, while the mitochondrion was strongly affected in parasites depleted with GGT-1. Importantly, depletion of GGT-2 caused the stronger defect to the sorting of rhoptry protein and the parasite morphology. Furthermore, parasite motility was observed to be affected in parasites depleted with GGT-2. Taken together, this study functionally characterized the prenyl transferases, which contributed to an overall understanding of protein prenylation in T. gondii and potentially in other related parasites. Full article

(This article belongs to the Special Issue CRISPR-Cas in Genomic Manipulation and Antimicrobial Resistance)

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11 pages, 1048 KiB

Open AccessArticle

Molecular Capture of Mycobacterium tuberculosis Genomes Directly from Clinical Samples: A Potential Backup Approach for Epidemiological and Drug Susceptibility Inferences

by Rita Macedo, Joana Isidro, Rita Ferreira, Miguel Pinto, Vítor Borges, Sílvia Duarte, Luís Vieira and João Paulo Gomes

Int. J. Mol. Sci. 2023, 24(3), 2912; https://doi.org/10.3390/ijms24032912 - 02 Feb 2023

Cited by 1 | Viewed by 1711

Abstract

The application of whole genome sequencing of Mycobacterium tuberculosis directly on clinical samples has been investigated as a means to avoid the time-consuming need for culture isolation that can lead to a potential prolonged suboptimal antibiotic treatment. We aimed to provide a proof-of-concept regarding the application of the molecular capture of M. tuberculosis genomes directly from positive sputum samples as an approach for epidemiological and drug susceptibility predictions. Smear-positive sputum samples (n = 100) were subjected to the SureSelectXT HS Target Enrichment protocol (Agilent Technologies, Santa Clara, CA, USA) and whole-genome sequencing analysis. A higher number of reads on target were obtained for higher smear grades samples (i.e., 3+ followed by 2+). Moreover, 37 out of 100 samples showed ≥90% of the reference genome covered with at least 10-fold depth of coverage (27, 9, and 1 samples were 3+, 2+, and 1+, respectively). Regarding drug-resistance/susceptibility prediction, for 42 samples, ≥90% of the >9000 hits that are surveyed by TB-profiler were detected. Our results demonstrated that M. tuberculosis genome capture and sequencing directly from clinical samples constitute a potential valid backup approach for phylogenetic inferences and resistance prediction, essentially in settings when culture is not routinely performed or for samples that fail to grow. Full article

(This article belongs to the Special Issue CRISPR-Cas in Genomic Manipulation and Antimicrobial Resistance)

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15 pages, 2373 KiB

Open AccessArticle

DNA Motifs and an Accessory CRISPR Factor Determine Cas1 Binding and Integration Activity in Sulfolobus islandicus

by Tao Liu, Ying Xu, Xiaojie Wang, Qing Ye, Zhenzhen Liu, Zhufeng Zhang, Jilin Liu, Yudong Yang, Xu Peng and Nan Peng

Int. J. Mol. Sci. 2022, 23(17), 10178; https://doi.org/10.3390/ijms231710178 - 05 Sep 2022

Cited by 1 | Viewed by 1512

Abstract

CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the leader-proximal end remains largely unknown. Here, in the Sulfolobus islandicus subtype I-A system, we show that mutations destroying the proximal region reduce CRISPR adaptation in vivo. We identify that a stem-loop structure is present on the leader-proximal end, and we demonstrate that Cas1 preferentially binds the stem-loop structure in vitro. Moreover, we demonstrate that the integrase activity of Cas1 is modulated by interacting with a CRISPR-associated factor Csa3a. When translocated to the CRISPR array, the Csa3a-Cas1 complex is separated by Csa3a binding to the leader-distal motif and Cas1 binding to the leader-proximal end. Mutation at the leader-distal motif reduces CRISPR adaptation efficiency, further confirming the in vivo function of leader-distal motif. Together, our results suggest a general model for binding of Cas1 protein to a leader motif and modulation of integrase activity by an accessory factor. Full article

(This article belongs to the Special Issue CRISPR-Cas in Genomic Manipulation and Antimicrobial Resistance)

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16 pages, 2637 KiB

Open AccessArticle

Inactivation of Target RNA Cleavage of a III-B CRISPR-Cas System Induces Robust Autoimmunity in Saccharolobus islandicus

by Yan Zhang, Jinzhong Lin, Xuhui Tian, Yuan Wang, Ruiliang Zhao, Chenwei Wu, Xiaoning Wang, Pengpeng Zhao, Xiaonan Bi, Zhenxiao Yu, Wenyuan Han, Nan Peng, Yun Xiang Liang and Qunxin She

Int. J. Mol. Sci. 2022, 23(15), 8515; https://doi.org/10.3390/ijms23158515 - 31 Jul 2022

Cited by 2 | Viewed by 1643

Abstract

Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in Saccharolobus islandicus, and this revealed that Cmr4α RNase-dead (dCmr4α) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4α in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4α inhibition. Expression of dCmr4α also strongly inhibited plasmid transformation with Cmr2α^HD and Cmr2α^Palm mutants, but the inhibition was diminished in Cmr2α^HD,Palm. Since dCmr4α-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-α system in the S. islandicus cells expressing dCmr4α. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems. Full article

(This article belongs to the Special Issue CRISPR-Cas in Genomic Manipulation and Antimicrobial Resistance)

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