Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems

2020 ◽  
Vol 89 (1) ◽  
pp. 309-332 ◽  
Author(s):  
Alan R. Davidson ◽  
Wang-Ting Lu ◽  
Sabrina Y. Stanley ◽  
Jingrui Wang ◽  
Marios Mejdani ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mobile Genetic Elements ◽  
Protein Inhibitors ◽  
Practical Applications ◽  
Viral Genomes ◽  
Molecular Detail ◽  
Foreign Dna ◽  
Adaptive Ability ◽  
Functional Mechanisms ◽  
Insight Into

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.

scholarly journals Structural insight into multistage inhibition of CRISPR-Cas12a by AcrVA4

2019 ◽  
Vol 116 (38) ◽  
pp. 18928-18936 ◽  
Author(s):  
Ruchao Peng ◽  
Zhiteng Li ◽  
Ying Xu ◽  
Shaoshuai He ◽  
Qi Peng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mobile Genetic Elements ◽  
Human Cells ◽  
Type I ◽  
Enzyme Recycling ◽  
Target Dna ◽  
Regulatory Genome ◽  
Structural Insight ◽  
Underlying Mechanisms ◽  
Insight Into

Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Lachnospiraceae bacterium Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.

scholarly journals Reovirus Low-Density Particles Package Cellular RNA

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1096
Author(s):  
Timothy W. Thoner ◽  
Xiang Ye ◽  
John Karijolich ◽  
Kristen M. Ogden
Keyword(s):  
Rna Polymerase ◽  
Viral Proteins ◽  
Viral Rna ◽  
Low Density ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Double Stranded Rna ◽  
Viral Genomes ◽  
Mammalian Orthoreovirus ◽  
Insight Into

Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and “genomeless” top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.

scholarly journals Draft Genome of Shewanella frigidimarina Ag06-30, a Marine Bacterium Isolated from Potter Peninsula, King George Island, Antarctica

2016 ◽  
Vol 4 (3) ◽  
Author(s):  
Gisela Parmeciano Di Noto ◽  
Susana C. Vázquez ◽  
Walter P. MacCormack ◽  
Andrés Iriarte ◽  
Cecilia Quiroga
Keyword(s):  
Gene Transfer ◽  
Lateral Gene Transfer ◽  
Marine Bacterium ◽  
Draft Genome ◽  
Mobile Genetic Elements ◽  
King George Island ◽  
Genetic Elements ◽  
Shewanella Frigidimarina ◽  
Insight Into ◽  
Potter Peninsula

We present the draft genome of Shewanella frigidimarina Ag06-30, a marine bacterium from King George Island, Antarctica, which encodes the carbapenemase SFP-1. The assembly contains 4,799,218 bp (G+C content 41.24%). This strain harbors several mobile genetic elements that provide insight into lateral gene transfer and bacterial plasticity and evolution.

Market Opportunity Analysis in Thailand: Case of Individual Power Sources by Thermoelectric-Generator Technology for Portable Electronics

2019 ◽  
Vol 16 (03) ◽  
pp. 1950027
Author(s):  
Surapree Maolikul ◽  
Thira Chavarnakul ◽  
Somchai Kiatgamolchai
Keyword(s):  
Thermoelectric Generator ◽  
Electrical Power ◽  
Technology Commercialization ◽  
Market Opportunity ◽  
Business Research ◽  
Power Sources ◽  
Practical Applications ◽  
Portable Electronics ◽  
Decision Factors ◽  
Insight Into

Thermoelectrics, an energy-conversion technology, has been developed for its potential to support portable electronics with an innovative power source. Primarily focusing on the metropolitan market in Thailand, the study, thus, aimed at the market insight into portable electronics users’ characteristics and opinions of thermoelectric-generator (TEG) technology commercialization. The business research was conducted to analyze their behaviors for power-supply lacking problems, encountering heat or cold sources, purchasing decision for a TEG-based charger and key decision factors. For practical applications, an innovative TEG-based charger should be more flexible by harnessing various heat or cold sources from ambient situations to generate electrical power.

Improving Techniques in Statically Equivalent Serial Chain Modeling for Center of Mass Estimation

2014 ◽  
Author(s):  
Bingjue Li ◽  
Andrew P. Murray ◽  
David H. Myszka
Keyword(s):  
Center Of Mass ◽  
Original System ◽  
Rigid Bodies ◽  
Joint Angles ◽  
Practical Applications ◽  
Serial Chain ◽  
System Of Rigid Bodies ◽  
Final Development ◽  
Data Error ◽  
Insight Into

Any articulated system of rigid bodies defines a Statically Equivalent Serial Chain (SESC). The SESC is a virtual chain that terminates at the center of mass (CoM) of the original system of bodies. A SESC may be generated experimentally without knowing the mass, CoM, or length of each link in the system given that its joint angles and overall CoM may be measured. This paper presents three developments toward recognizing the SESC as a practical modeling technique. Two of the three developments improve utilizing the technique in practical applications where the arrangement of the joints impacts the derivation of the SESC. The final development provides insight into the number of poses needed to create a usable SESC in the presence of data collection errors. First, modifications to a matrix necessary in computing the SESC are proposed. Second, the problem of generating a SESC experimentally when the system of bodies includes a mass fixed in the ground frame are presented and a remedy is proposed for humanoid-like systems. Third, an investigation of the error of the experimental SESC versus the number of data readings collected in the presence of errors in joint readings and CoM data is conducted. By conducting the method on three different systems with various levels of data error, a general form of the function for estimating the error of the experimental SESC is proposed.

scholarly journals Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

2021 ◽  
Author(s):  
Bijoya Paul ◽  
Loic Chaubet ◽  
Emma Verver ◽  
Guillermo Montoya
Keyword(s):  
Dna Binding ◽  
Genome Editing ◽  
Optical Tweezers ◽  
Target Site ◽  
Target Dna ◽  
Multiple Binding ◽  
Target Sites ◽  
Dna Binding And Cleavage ◽  
Target Activity ◽  
Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

The Temporal and the Eternal

2005 ◽  
pp. 611-636
Keyword(s):  
Rational Decision ◽  
Practical Applications ◽  
The World ◽  
The Universe ◽  
Insight Into

This chapter, taken from Josiah Royce's Gifford Lectures of 1899, argues that any rational decision as between a pessimistic and an optimistic view of the world, any account of the relations between God and Man, any view of the sense in which the evils and imperfections of the Universe can be comprehended or justified, any account of our ethical consciousness in terms reconcilable with our Idealism must turn in part upon a distinction between the Temporal and the Eternal, and upon an insight into their unity in the midst of their contrast. The problem at issue is one of the most delicate and, at the same time, one of the simplest of the great issues of philosophy. The chapter deals with it at first in a purely theoretical fashion and then proceeds to its practical applications.

Literature Review of Clergy Resilience and Recommendations for Future Research

2020 ◽  
pp. 009164712096813
Author(s):  
Andrea M. Sielaff ◽  
Kate Rae Davis ◽  
J. Derek McNeil
Keyword(s):  
Literature Review ◽  
Traumatic Stress ◽  
Well Being ◽  
Work Life ◽  
Future Research ◽  
Review Of The Literature ◽  
Practical Applications ◽  
Insight Into ◽  
Professional Ministry

Clergy often experience a call to help others; however, this passion is hard to sustain because of the chronic and traumatic stress that are components of the job. Because of the unique stressors that are part of professional ministry, clergy need targeted support that is systemic as well as individual to practice resilience. This review of the research provides insight into what factors most impact clergy well-being; as congregations, supervisors, and denominations learn more about these factors, they can more effectively create environments in which clergy can be resilient. This review of the literature also illuminates what clergy might do for themselves to create a sustainable work life that supports their growth and thriving in the midst of adversity in ministry. In addition to articulating the specific stressors clergy face, this review resources congregations, clergy, and supervisors with practical applications of resilience research.

scholarly journals Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
April Pawluk ◽  
Megha Shah ◽  
Marios Mejdani ◽  
Charles Calmettes ◽  
Trevor F. Moraes ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Repressor ◽  
Mobile Genetic Elements ◽  
Type I ◽  
Genetic Studies ◽  
Genetic Elements ◽  
Crispr System ◽  
Mechanistic Insight ◽  
Resistance To Invasion ◽  
Insight Into

ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system.

scholarly journals Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells

2020 ◽  
Vol 11 ◽  
Author(s):  
Maor Sheva ◽  
Uri Hanania ◽  
Tami Ariel ◽  
Albina Turbovski ◽  
Vishal Kumar Rameshchandra Rathod ◽  
...  
Keyword(s):  
Host Cell ◽  
Genome Editing ◽  
Cell Lines ◽  
Recombinant Proteins ◽  
Plant Cells ◽  
Second Step ◽  
Selectable Markers ◽  
Additional Gene ◽  
Bright Yellow ◽  
Foreign Dna

While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery. In this study we developed an innovative stepwise methodology in which the CRISPR-Cas9 is used sequentially to target genome editing, followed by its own excision. The first step included a stable insertion of a CRISPR-Cas9 cassette, targeted to knockout the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. The second step included the excision of the inserted cassette of 14.3 kbp by induction of specific sgRNA designed to target the T-DNA boundaries. The genome editing step and the transgene removal step are achieved in one transformation run. This mechanism enables CRISPR genome editing and subsequently eliminating the introduced transgenes thus freeing the cells from foreign DNA no longer needed.

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