scholarly journals New and emerging uses of CRISPR/Cas9 to genetically manipulate apicomplexan parasites

Parasitology ◽  
2018 ◽  
Vol 145 (9) ◽  
pp. 1119-1126 ◽  
Author(s):  
Manlio Di Cristina ◽  
Vern B. Carruthers
Keyword(s):  
Toxoplasma Gondii ◽  
Genome Engineering ◽  
Gene Knockout ◽  
Full Potential ◽  
Whole Genome ◽  
Apicomplexan Parasites ◽  
Genetic Manipulations ◽  
Knockout Mutants ◽  
History Of ◽  
Genome Screening

AbstractAlthough the application of CRISPR/Cas9 genome engineering approaches was first reported in apicomplexan parasites only 3 years ago, this technology has rapidly become an essential component of research on apicomplexan parasites. This review briefly describes the history of CRISPR/Cas9 and the principles behind its use along with documenting its implementation in apicomplexan parasites, especially Plasmodium spp. and Toxoplasma gondii. We also discuss the recent use of CRISPR/Cas9 for whole genome screening of gene knockout mutants in T. gondii and highlight its use for seminal genetic manipulations of Cryptosporidium spp. Finally, we consider new variations of CRISPR/Cas9 that have yet to be implemented in apicomplexans. Whereas CRISPR/Cas9 has already accelerated rapid interrogation of gene function in apicomplexans, the full potential of this technology is yet to be realized as new variations and innovations are integrated into the field.

scholarly journals Generation and Application of a Versatile CRISPR Toolkit for Mammalian Cell Engineering

2021 ◽  
Author(s):  
Nghi Dang ◽  
Alissa Lance-Byrne ◽  
Angela Tung ◽  
Xiaoge Guo ◽  
Ryan J Cecchi ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Genome Engineering ◽  
Gene Knockout ◽  
Transient Transfection ◽  
Cell Engineering ◽  
Genetic Manipulations ◽  
Expression Plasmids

Abstract CRISPR/Cas9 has revolutionized the field of genome engineering. Yet, as the CRISPR toolbox has rapidly expanded, there remains a need for a comprehensive library of CRISPR/Cas9 reagents that allow users to perform complex cellular and genetic manipulations without requiring labor-intensive generation of reagents to meet each user’s unique experimental circumstances. Here we described the creation and validation of a pNAX CRISPR library consisting of 72 different Cas9 and gRNA expression plasmids to allow for efficient multiplex gene editing, activation, and repression in mammalian cells. The toolkit plasmids, which are piggyBac or lentiviral based, provide the means for reliable and rapid delivery of Cas9/gRNA through either transient transfection or stable integration. Using the toolkit, we demonstrate the ease with which users can perform single or multiplex gene editing and modulate the expression of both coding and non-coding genes. We also highlight the use of the comprehensive toolkit to perform combinatorial gene knockout to identify factors that regulate homologous recombination, along with investigating the regulatory role of a 68-kb intronic region associated with human disease.

scholarly journals Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 476
Author(s):  
Joachim Kloehn ◽  
Matteo Lunghi ◽  
Emmanuel Varesio ◽  
David Dubois ◽  
Dominique Soldati-Favre
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Rna Degradation ◽  
Major Facilitator Superfamily ◽  
Untargeted Metabolomics ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Major Facilitator ◽  
Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

Phylogenetic Analysis of T-Box Genes Demonstrates the Importance of Amphioxus for Understanding Evolution of the Vertebrate Genome

Genetics ◽  
2000 ◽  
Vol 156 (3) ◽  
pp. 1249-1257
Author(s):  
Ilya Ruvinsky ◽  
Lee M Silver ◽  
Jeremy J Gibson-Brown
Keyword(s):  
Phylogenetic Analysis ◽  
Whole Genome Duplication ◽  
Genome Duplication ◽  
Gene Families ◽  
Vertebrate Evolution ◽  
Whole Genome ◽  
Vertebrate Genome ◽  
T Box ◽  
Genetic Complexity ◽  
History Of

Abstract The duplication of preexisting genes has played a major role in evolution. To understand the evolution of genetic complexity it is important to reconstruct the phylogenetic history of the genome. A widely held view suggests that the vertebrate genome evolved via two successive rounds of whole-genome duplication. To test this model we have isolated seven new T-box genes from the primitive chordate amphioxus. We find that each amphioxus gene generally corresponds to two or three vertebrate counterparts. A phylogenetic analysis of these genes supports the idea that a single whole-genome duplication took place early in vertebrate evolution, but cannot exclude the possibility that a second duplication later took place. The origin of additional paralogs evident in this and other gene families could be the result of subsequent, smaller-scale chromosomal duplications. Our findings highlight the importance of amphioxus as a key organism for understanding evolution of the vertebrate genome.

scholarly journals Resistome Profiles, Plasmid Typing, and Whole-Genome Phylogenetic Tree Analyses of BlaNDM-9 and Mcr-1 Co-Harboring Escherichia coli ST617 from a Patient without a History of Farm Exposure in Korea

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 212 ◽  
Author(s):  
Le Phuong Nguyen ◽  
Naina Adren Pinto ◽  
Thao Nguyen Vu ◽  
Hung Mai ◽  
An HT Pham ◽  
...  
Keyword(s):  
Rectal Swab ◽  
Asymptomatic Carrier ◽  
University Hospital ◽  
Surveillance Culture ◽  
Whole Genome ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Plasmid Replicon ◽  
Donor Cells ◽  
History Of

Recently, a blaNDM-9 and mcr-1 co-harboring E. coli ST 617 isolate was identified from an asymptomatic carrier in Korea. An 81-year-old female was admitted to a university hospital for aortic cardiac valve repair surgery. Following surgery, she was admitted to the intensive care unit (ICU) for three days, and carbapenem-resistant E. coli YMC/2017/02/MS631 was isolated from a surveillance culture (rectal swab). Antimicrobial susceptibility testing (AST) for colistin was not performed at that time. Upon retrospective study, further AST revealed resistance to all tested antibiotics, including meropenem, imipenem, ceftazidime-avibactam, amikacin, gentamicin, ciprofloxacin, trimethoprim-sulfamethoxazole, and colistin, with the exception of tigecycline. Whole genome sequencing analyses showed that this strain belonged to the ST617 serotype O89/162: H10 and harbored three β-lactamase genes (blaTEM-1B, blaCTX-M-55, blaNDM-9), mcr-1, and 14 other resistance genes. Seven plasmid replicon types (IncB, IncFII, IncI2, IncN, IncY, IncR, IncX1) were identified. Horizontal transfer of blaNDM-9 and mcr-1 from donor cells to the recipient E. coli J53 has been observed. blaNDM-9 and mcr-1 were carried by IncB and IncI2 plasmids, respectively. To speculate on the incidence of this strain, routine rectal swab screening to identify asymptomatic carriers might be warranted, in addition to the screening of ICU patients.

scholarly journals Analysis of whole-genome re-sequencing data of ducks reveals a diverse demographic history and extensive gene flow between Southeast/South Asian and Chinese populations

2021 ◽  
Vol 53 (1) ◽  
Author(s):  
Fan Jiang ◽  
Ruiyi Lin ◽  
Changyi Xiao ◽  
Tanghui Xie ◽  
Yaoxin Jiang ◽  
...  
Keyword(s):  
Gene Flow ◽  
South Asia ◽  
South Asian ◽  
Demographic History ◽  
Nervous System Development ◽  
Chromosome 1 ◽  
Whole Genome ◽  
Chinese Populations ◽  
Asian Populations ◽  
History Of

Abstract Background The most prolific duck genetic resource in the world is located in Southeast/South Asia but little is known about the domestication and complex histories of these duck populations. Results Based on whole-genome resequencing data of 78 ducks (Anas platyrhynchos) and 31 published whole-genome duck sequences, we detected three geographic distinct genetic groups, including local Chinese, wild, and local Southeast/South Asian populations. We inferred the demographic history of these duck populations with different geographical distributions and found that the Chinese and Southeast/South Asian ducks shared similar demographic features. The Chinese domestic ducks experienced the strongest population bottleneck caused by domestication and the last glacial maximum (LGM) period, whereas the Chinese wild ducks experienced a relatively weak bottleneck caused by domestication only. Furthermore, the bottleneck was more severe in the local Southeast/South Asian populations than in the local Chinese populations, which resulted in a smaller effective population size for the former (7100–11,900). We show that extensive gene flow has occurred between the Southeast/South Asian and Chinese populations, and between the Southeast Asian and South Asian populations. Prolonged gene flow was detected between the Guangxi population from China and its neighboring Southeast/South Asian populations. In addition, based on multiple statistical approaches, we identified a genomic region that included three genes (PNPLA8, THAP5, and DNAJB9) on duck chromosome 1 with a high probability of gene flow between the Guangxi and Southeast/South Asian populations. Finally, we detected strong signatures of selection in genes that are involved in signaling pathways of the nervous system development (e.g., ADCYAP1R1 and PDC) and in genes that are associated with morphological traits such as cell growth (e.g., IGF1R). Conclusions Our findings provide valuable information for a better understanding of the domestication and demographic history of the duck, and of the gene flow between local duck populations from Southeast/South Asia and China.

scholarly journals An Investigation of the Glucose Monitoring Practices of Nurses in Stroke Care: A Descriptive Cohort Study

2013 ◽  
Vol 2013 ◽  
pp. 1-7
Author(s):  
Elizabeth Ann Laird ◽  
Vivien E. Coates ◽  
Assumpta A. Ryan ◽  
Mark O. McCarron ◽  
Diane Lyttle ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Cohort Study ◽  
Acute Stroke ◽  
Glucose Monitoring ◽  
Positive Association ◽  
Stroke Care ◽  
Routine Practice ◽  
Full Potential ◽  
Significant Positive Association ◽  
History Of

Glucose derangement is commonly observed among adults admitted to hospital with acute stroke. This paper presents the findings from a descriptive cohort study that investigated the glucose monitoring practices of nurses caring for adults admitted to hospital with stroke or transient ischaemic attack. We found that a history of diabetes mellitus was strongly associated with initiation of glucose monitoring and higher frequency of that monitoring. Glucose monitoring was continued for a significantly longer duration of days for adults with a history of diabetes mellitus, when compared to the remainder of the cohort. As glucose monitoring was not routine practice for adults with no history of diabetes mellitus, the detection and treatment of hyperglycaemia and hypoglycaemia events could be delayed. There was a significant positive association between the admission hospital that is most likely to offer stroke unit care and the opportunity for glucose monitoring. We concluded that adults with acute stroke, irrespective of their diabetes mellitus status prior to admission to hospital, are vulnerable to both hyperglycaemic and hypoglycaemic events. This study suggests that the full potential of nurses in the monitoring of glucose among hospitalised adults with stroke has yet to be realised.

Whole Genome Screening for Sick Newborns: Equity Now

2021 ◽  
Vol 8 (6) ◽  
pp. 3-3
Author(s):  
Sharon F. Terry
Keyword(s):  
Whole Genome ◽  
Genome Screening

scholarly journals Toxoplasma gondii myosins B/C

2001 ◽  
Vol 155 (4) ◽  
pp. 613-624 ◽  
Author(s):  
Frédéric Delbac ◽  
Astrid Sänger ◽  
Eva M. Neuhaus ◽  
Rolf Stratmann ◽  
James W. Ajioka ◽  
...  
Keyword(s):  
Cell Division ◽  
Toxoplasma Gondii ◽  
Daughter Cell ◽  
Gliding Motility ◽  
Apicomplexan Parasites ◽  
Daughter Cells ◽  
Membrane Complex ◽  
Alternatively Spliced ◽  
Butanedione Monoxime ◽  
Inner Membrane Complex

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

scholarly journals The Plastid of Toxoplasma gondii Is Divided by Association with the Centrosomes

2000 ◽  
Vol 151 (7) ◽  
pp. 1423-1434 ◽  
Author(s):  
Boris Striepen ◽  
Michael J. Crawford ◽  
Michael K. Shaw ◽  
Lewis G. Tilney ◽  
Frank Seeber ◽  
...  
Keyword(s):  
Cell Division ◽  
Toxoplasma Gondii ◽  
Genetic Manipulation ◽  
Fluorescent Proteins ◽  
Recombinant Expression ◽  
Molecular Genetic ◽  
Time Lapse ◽  
Microtubule Organization ◽  
Apicomplexan Parasites ◽  
Daughter Cells

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.

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