scholarly journals History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology

2018 ◽  
Vol 200 (7) ◽  
Author(s):  
Yoshizumi Ishino ◽  
Mart Krupovic ◽  
Patrick Forterre
Keyword(s):  
Genome Editing ◽  
Functional Characterization ◽  
Halophilic Archaea ◽  
Repeated Sequence ◽  
Hyperthermophilic Archaea ◽  
Content Type ◽  
Origin And Evolution ◽  
History Of ◽  
Short Palindromic Repeat ◽  
Original Observation

ABSTRACTClustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are well-known acquired immunity systems that are widespread in archaea and bacteria. The RNA-guided nucleases from CRISPR-Cas systems are currently regarded as the most reliable tools for genome editing and engineering. The first hint of their existence came in 1987, when an unusual repetitive DNA sequence, which subsequently was defined as a CRISPR, was discovered in theEscherichia coligenome during an analysis of genes involved in phosphate metabolism. Similar sequence patterns were then reported in a range of other bacteria as well as in halophilic archaea, suggesting an important role for such evolutionarily conserved clusters of repeated sequences. A critical step toward functional characterization of the CRISPR-Cas systems was the recognition of a link between CRISPRs and the associated Cas proteins, which were initially hypothesized to be involved in DNA repair in hyperthermophilic archaea. Comparative genomics, structural biology, and advanced biochemistry could then work hand in hand, not only culminating in the explosion of genome editing tools based on CRISPR-Cas9 and other class II CRISPR-Cas systems but also providing insights into the origin and evolution of this system from mobile genetic elements denoted casposons. To celebrate the 30th anniversary of the discovery of CRISPR, this minireview briefly discusses the fascinating history of CRISPR-Cas systems, from the original observation of an enigmatic sequence inE. colito genome editing in humans.

scholarly journals Evolution of the U.S. Biological Select AgentRathayibacter toxicus

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Edward W. Davis ◽  
Javier F. Tabima ◽  
Alexandra J. Weisberg ◽  
Lucas Dantas Lopes ◽  
Michele S. Wiseman ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Adaptive Immune System ◽  
The United States ◽  
Genomic Diversity ◽  
Data Set ◽  
Important Species ◽  
Content Type ◽  
Low Genetic Diversity ◽  
History Of ◽  
Short Palindromic Repeat

ABSTRACTRathayibacter toxicusis a species of Gram-positive, corynetoxin-producing bacteria that causes annual ryegrass toxicity, a disease often fatal to grazing animals. A phylogenomic approach was employed to model the evolution ofR. toxicusto explain the low genetic diversity observed among isolates collected during a 30-year period of sampling in three regions of Australia, gain insight into the taxonomy ofRathayibacter, and provide a framework for studying these bacteria. Analyses of a data set of more than 100 sequencedRathayibactergenomes indicated thatRathayibacterforms nine species-level groups.R. toxicusis the most genetically distant, and evidence suggested that this species experienced a dramatic event in its evolution. Its genome is significantly reduced in size but is colinear to those of sister species. Moreover,R. toxicushas low intergroup genomic diversity and almost no intragroup genomic diversity between ecologically separated isolates.R. toxicusis the only species of the genus that encodes a clustered regularly interspaced short palindromic repeat (CRISPR) locus and that is known to host a bacteriophage parasite. The spacers, which represent a chronological history of infections, were characterized for information on past events. We propose a three-stage process that emphasizes the importance of the bacteriophage and CRISPR in the genome reduction and low genetic diversity of theR. toxicusspecies.IMPORTANCERathayibacter toxicusis a toxin-producing species found in Australia and is often fatal to grazing animals. The threat of introduction of the species into the United States led to its inclusion in the Federal Select Agent Program, which makesR. toxicusa highly regulated species. This work provides novel insights into the evolution ofR. toxicus.R. toxicusis the only species in the genus to have acquired a CRISPR adaptive immune system to protect against bacteriophages. Results suggest that coexistence with the bacteriophage NCPPB3778 led to the massive shrinkage of theR. toxicusgenome, species divergence, and the maintenance of low genetic diversity in extant bacterial groups. This work contributes to an understanding of the evolution and ecology of an agriculturally important species of bacteria.

scholarly journals A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Mei-Yi Yan ◽  
Si-Shang Li ◽  
Xin-Yuan Ding ◽  
Xiao-Peng Guo ◽  
Qi Jin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genome Editing ◽  
Large Scale ◽  
Nonhomologous End Joining ◽  
Genetic Mutations ◽  
Repair Pathway ◽  
End Joining ◽  
Content Type ◽  
Nhej Repair ◽  
Short Palindromic Repeat

ABSTRACT New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis. Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis. Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli. IMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis. More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.

scholarly journals Variation of the Virus-Related Elements within Syntenic Genomes of the Hyperthermophilic Archaeon Aeropyrum

2013 ◽  
Vol 79 (19) ◽  
pp. 5891-5898 ◽  
Author(s):  
Takashi Daifuku ◽  
Takashi Yoshida ◽  
Takayuki Kitamura ◽  
Satoshi Kawaichi ◽  
Takahiro Inoue ◽  
...  
Keyword(s):  
Gc Content ◽  
Adaptation Strategy ◽  
Hyperthermophilic Archaea ◽  
Orthologous Genes ◽  
Entire Genome ◽  
Content Type ◽  
Aeropyrum Pernix ◽  
System A ◽  
Genome Features ◽  
Short Palindromic Repeat

ABSTRACTThe increasing number of genome sequences of archaea and bacteria show their adaptation to different environmental conditions at the genomic level.Aeropyrumspp. are aerobic and hyperthermophilic archaea.Aeropyrum caminiwas isolated from a deep-sea hydrothermal vent, andAeropyrum pernixwas isolated from a coastal solfataric vent. To investigate the adaptation strategy in each habitat, we compared the genomes of the two species. Shared genome features were a small genome size, a high GC content, and a large portion of orthologous genes (86 to 88%). The genomes also showed high synteny. These shared features may have been derived from the small number of mobile genetic elements and the lack of a RecBCD system, a recombinational enzyme complex. In addition, the specialized physiology (aerobic and hyperthermophilic) ofAeropyrumspp. may also contribute to the entire-genome similarity. Despite having stable genomes, interference of synteny occurred with two proviruses,A. pernixspindle-shaped virus 1 (APSV1) andA. pernixovoid virus 1 (APOV1), and clustered regularly interspaced short palindromic repeat (CRISPR) elements. Spacer sequences derived from theA. caminiCRISPR showed significant matches with protospacers of the two proviruses infectingA. pernix, indicating thatA. caminiinteracted with viruses closely related to APSV1 and APOV1. Furthermore, a significant fraction of the nonorthologous genes (41 to 45%) were proviral genes or ORFans probably originating from viruses. Although the genomes ofA. caminiandA. pernixwere conserved, we observed nonsynteny that was attributed primarily to virus-related elements. Our findings indicated that the genomic diversification ofAeropyrumspp. is substantially caused by viruses.

scholarly journals CRISPR-Cas12a-Assisted Recombineering in Bacteria

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Mei-Yi Yan ◽  
Hai-Qin Yan ◽  
Gai-Xian Ren ◽  
Ju-Ping Zhao ◽  
Xiao-Peng Guo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mycobacterium Smegmatis ◽  
Genetic Manipulation ◽  
Antibiotic Resistance Gene ◽  
Point Mutations ◽  
Content Type ◽  
Short Palindromic Repeat ◽  
New Type ◽  
System Point ◽  
Selection Of

ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis. Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria. IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.

scholarly journals Novel SCN5A p.Val1667Asp Missense Variant Segregation and Characterization in a Family with Severe Brugada Syndrome and Multiple Sudden Deaths

2021 ◽  
Vol 22 (9) ◽  
pp. 4700
Author(s):  
Michelle M. Monasky ◽  
Emanuele Micaglio ◽  
Giuseppe Ciconte ◽  
Ilaria Rivolta ◽  
Valeria Borrelli ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Family History ◽  
Risk Stratification ◽  
Brugada Syndrome ◽  
Electrophysiological Study ◽  
Functional Characterization ◽  
The Family ◽  
Novel Variant ◽  
History Of ◽  
Scn5a Gene

Genetic testing in Brugada syndrome (BrS) is still not considered to be useful for clinical management of patients in the majority of cases, due to the current lack of understanding about the effect of specific variants. Additionally, family history of sudden death is generally not considered useful for arrhythmic risk stratification. We sought to demonstrate the usefulness of genetic testing and family history in diagnosis and risk stratification. The family history was collected for a proband who presented with a personal history of aborted cardiac arrest and in whom a novel variant in the SCN5A gene was found. Living family members underwent ajmaline testing, electrophysiological study, and genetic testing to determine genotype-phenotype segregation, if any. Patch-clamp experiments on transfected human embryonic kidney 293 cells enabled the functional characterization of the SCN5A novel variant in vitro. In this study, we provide crucial human data on the novel heterozygous variant NM_198056.2:c.5000T>A (p.Val1667Asp) in the SCN5A gene, and demonstrate its segregation with a severe form of BrS and multiple sudden deaths. Functional data revealed a loss of function of the protein affected by the variant. These results provide the first disease association with this variant and demonstrate the usefulness of genetic testing for diagnosis and risk stratification in certain patients. This study also demonstrates the usefulness of collecting the family history, which can assist in understanding the severity of the disease in certain situations and confirm the importance of the functional studies to distinguish between pathogenic mutations and harmless genetic variants.

A tribute to Gwen Leighty

2006 ◽  
Vol 19 (2) ◽  
pp. 84-86
Author(s):  
Jennifer Paustenbaugh
Keyword(s):  
Web Sites ◽  
Academic Libraries ◽  
Design Methodology ◽  
Fund Raising ◽  
Personal Knowledge ◽  
Valuable Contribution ◽  
Content Type ◽  
History Of ◽  
Development Network ◽  
Fund Raiser

PurposeThe purpose of the paper is to provide a tribute to the life and work of library fund‐raiser Gwen Leighty.Design/methodology/approachThe paper uses personal knowledge and references to Academic Libraries Advancement and Development Network (ALADN) and LIBDEV web sites.FindingsThe paper finds that fundraising is connecting with people and the journey that each development officer must make while raising funds for their library.Originality/valueThe paper presents a brief history of ALADN and the valuable contribution one person made to the cause of library fund‐raising.

The sediment of the Hostivar Reservoir (Prague, Czech Republic) as a memory of 45 years of pollution

2017 ◽  
Vol 28 (2) ◽  
pp. 204-213
Author(s):  
Lucie Soucková ◽  
Dana Kominkova
Keyword(s):  
Czech Republic ◽  
Design Methodology ◽  
Polyaromatic Hydrocarbons ◽  
Reservoir Sediment ◽  
Terrestrial Environment ◽  
Content Type ◽  
Sediment Extraction ◽  
History Of ◽  
Us Epa ◽  
Practical Implications

Purpose The purpose of this paper is to evaluate the historical pollution of the Hostivar Reservoir (largest reservoir in Prague) sediment by metals, polyaromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCB) and identify the trends in pollution of aquatic environment. Design/methodology/approach Core samples, 140 cm long, recording the 45-year history of the reservoir, were separated to 5 cm width subsamples (approximately 1.5 years of sedimentation) and analyzed for metals (Cd, Pb, Cu, Zn, Cr, Ni, Al), PAH and PCB. Following methods were used: US EPA 3051 for metals, US EPA 505 and US EPA 8082 A for PCB, and ISO 18287:2006 for PAH. Findings Most of the contaminants had the highest concentration at the beginning of the existence of the reservoir, suggesting that the contamination results from construction activities. Significant decrease of Pb occurred in the second half of the 1990s. It was caused by termination of the addition of lead as a detonation suppressant to the gasoline. Most concentrations of PAHs, PCBs and metals, except copper do not present eco-toxicological risk. Practical implications The results show the volume of priority pollutants removed from the reservoir by sediment extraction, and point risk to the terrestrial environment due to application of the sediment in the construction of a noise protecting wall. Originality/value The paper presents unique data about historical contamination of the largest reservoir in Prague, the capital of Czech Republic. It shows how the watershed and the construction phase of the dam cause a pollution of the reservoir sediment and possible environmental risk for aquatic biota.

A brief history of small business in Australia, 1970-2010

2014 ◽  
Vol 3 (2) ◽  
pp. 222-236 ◽  
Author(s):  
Michael T. Schaper
Keyword(s):  
Small Business ◽  
Small Firms ◽  
Formal Education ◽  
Extensive Literature ◽  
Business Sector ◽  
Role Of Government ◽  
Content Type ◽  
Support Tools ◽  
History Of ◽  
Training And Research

Purpose – The purpose of this paper is to provide an overview of the development of the SME sector in Australia, concentrating on a number of key areas: small business definitions and numbers; the role of government; the emergence of key industry groups; and the evolution of education, training and research services. Design/methodology/approach – The study is a result of extensive literature reviews, desk research and the recollections of various participants in the field. Findings – There have been major changes to the Australian small business sector over the last 40 years. In 1983-1984 there were an estimated 550,000 small firms, and by 2010 this had grown to almost two million. Government involvement in, and support for, SMEs was virtually non-existent before 1970. Following the delivery of the Wiltshire report (1971), however, both state and federal governments responded by developing specialist advisory services, funding programmes and other support tools. Virtually non-existent before the 1970s, several peak industry associations were formed between 1977 and the 1990s. At the same time, formal education and teaching in the area expanded in the 1970s and 1980s and is now widespread. Practical implications – Development of the small business sector in Australia has often paralleled similar trends in other OECD nations. State and territory governments have often (but not always) been the principal drivers of policy change. Originality/value – There has been no little, if any, prior documentation of the evolution of the small business sector in Australia in the last 40 years.

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