scholarly journals The Versatile Type V CRISPR Effectors and Their Application Prospects

2021 ◽  
Vol 8 ◽  
Author(s):  
Baisong Tong ◽  
Huina Dong ◽  
Yali Cui ◽  
Pingtao Jiang ◽  
Zhaoxia Jin ◽  
...  
Keyword(s):  
Gene Editing ◽  
Effector Protein ◽  
Type Ii ◽  
Valuable Resource ◽  
Current Application ◽  
Convenient Tool ◽  
Type V ◽  
Development Prospects ◽  
Single Effector ◽  
Application Prospects

The class II clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems, characterized by a single effector protein, can be further subdivided into types II, V, and VI. The application of the type II CRISPR effector protein Cas9 as a sequence-specific nuclease in gene editing has revolutionized this field. Similarly, Cas13 as the effector protein of type VI provides a convenient tool for RNA manipulation. Additionally, the type V CRISPR–Cas system is another valuable resource with many subtypes and diverse functions. In this review, we summarize all the subtypes of the type V family that have been identified so far. According to the functions currently displayed by the type V family, we attempt to introduce the functional principle, current application status, and development prospects in biotechnology for all major members.

scholarly journals Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering for Cell Therapy Development

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3984-3984
Author(s):  
Gregory J. Cost ◽  
Rebecca C. Lamothe ◽  
Meghan D. Storlie ◽  
Andres Rivas ◽  
Diego A. Espinosa ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
High Frequency ◽  
Gene Editing ◽  
Constant Region ◽  
Type Ii ◽  
Novel Gene ◽  
Therapy Development ◽  
Type V

Abstract Gene-editing technology has revolutionized molecular therapeutics, enabling DNA-engineering-based approaches to treat disease. Despite this, development of medicines using gene editing has been hampered by technological, immunological, and legal limitations. We described previously the discovery of novel type II and type V gene-editing systems from metagenomic data, the characterization of these systems in vitro, and a demonstration of their activity in immortalized cell lines. Here, we substantially advance this work with three separate, novel gene-editing systems, demonstrating their utility for cell therapy development. We express and purify the nuclease components of both type II and type V effectors and show that all three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain constant region occurred in up to 95% of cells, both copies of the TCR beta-chain constant region in up to ~90% of cells, and beta-2 microglobulin in up to 95% of cells. Simultaneous double knock-out of TRAC and TRBC was obtained at an equal frequency. Gene editing with our systems had no effect on T cell viability. Further, we use our novel gene-editing systems to exploit homology-dependent DNA repair to integrate a CAR construct into the TCR alpha-chain locus (in up to ~60% of T cells), and demonstrate high-level CAR expression and antigen-dependent CAR-T cytotoxicity. Such robust editing activity at the TCR loci will permit efficient engineering and manufacture of allogeneic chimeric antigen receptor- (CAR) and TCR-based cell therapies. We next applied our novel gene-editing tools to NK cells and B cells. We achieved almost 100% gene disruption at the CD38 locus in NK cells and integrated a chimeric antigen receptor into the NK cell genome at a frequency of ~40%. Such CAR-NK cells displayed robust CAR-directed cellular cytotoxicity. B cell editing occurred in approximately 80% of target cells with successful transgene integration. Cas9 is notorious for tolerating mismatches and bulges in its target, resulting in high levels of unwanted DNA double-strand break formation. In contrast, interrogation of our gene editing systems using GUIDE-seq reveals no or very few off-target sites even at doses greatly in excess of those required for high-frequency gene editing. Finally, as our systems are taken from microbes found in environmental samples rather than from human pathogens, we expect pre-existing immunity to our nucleases will be quite rare. In all, we show that three new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development. Disclosures No relevant conflicts of interest to declare.

The distribution and density of ciliary tufts on the siphons of Anodonta cygnea (Mollusca: Bivalvia)

1990 ◽  
Vol 48 (3) ◽  
pp. 518-519
Author(s):  
Wen-lung Wu
Keyword(s):  
Osmium Tetroxide ◽  
Internal Surface ◽  
Mechanical Stimuli ◽  
Type Ii ◽  
Sensory Receptors ◽  
Anodonta Cygnea ◽  
Type Iv ◽  
Sem Study ◽  
Inhalant Siphon ◽  
Type V

The mantle of bivalves has come entirely to enclose the laterally compressed body and the mantle margin has assumed a variety of functions, one of the pricipal ones being sensory. Ciliary tufts, which are probably sensory, have been reported from the mantle and siphons of several bivalves1∽4. Certain regions of the mantle margin are likely to be more or less, sensitive to certain stimuli than others. The inhalant siphon is likely to be particularly sensitive to both chemical and mechanical stimuli, whereas the exhalant siphon will be less sensitive to both. The distribution and density of putative sensory receptors on the in-and ex-halant siphon is compared in this paper.The excised siphons were fixed in glutaraldehyde and osmium tetroxide, the whole procedure of SEM study is recorded in Wu's thesis.Type II cilia cover the tips of tentacles, 6.13um. Type IV and type V cilia are found on the surface of tentacles. Type IV cilia are occasionally present at the tips of tentacles, 8 um long. They are the commonest type on the surface of tentacles. Type VI cilia occor in the internal surface of the inhalant siphon, but are not found on the surface of tentacles, 6.7-10um long.

scholarly journals Subcellular Distribution and Relative Expression of Fibrocyte Markers in the CD/1 Mouse Cochlea Assessed by Semiquantitative Immunogold Electron Microscopy

2011 ◽  
Vol 59 (11) ◽  
pp. 984-1000 ◽  
Author(s):  
Shanthini Mahendrasingam ◽  
Catherine Bebb ◽  
Ella Shepard ◽  
David N. Furness
Keyword(s):  
Electron Microscopy ◽  
Tissue Growth ◽  
Immunogold Electron Microscopy ◽  
Spiral Ligament ◽  
Type Ii ◽  
Relative Expression ◽  
Aquaporin 1 ◽  
Cellular Transplantation ◽  
S 100 ◽  
Type V

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic. Here, we use immunogold electron microscopy to assess the relative expression of markers in native fibrocytes of the CD/1 mouse spiral ligament. Caldesmon and aquaporin 1 are expressed more in type III fibrocytes than any other type. S-100 is strongly expressed in types I, II, and V fibrocytes, and α1Na,K-ATPase is expressed strongly only in types II and V. By combining caldesmon or aquaporin 1 with S-100 and α1Na,K-ATPase, a ratiometric analysis of immunogold density distinguishes all except type II and type V fibrocytes. Other putative markers (creatine kinase BB and connective tissue growth factor) did not provide additional useful analytical attributes. By labeling serial sections or by double or triple labeling with combinations of three antibodies, this technique could be used to distinguish all except type II and type V fibrocytes in culture or after cellular transplantation into the lateral wall.

scholarly journals Native Gold in the Chudnoe Au-Pd-REE Deposit (Subpolar Urals, Russia): Composition, Minerals in Intergrowth and Genesis

Minerals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 451
Author(s):  
Galina Palyanova ◽  
Valery Murzin ◽  
Andrey Borovikov ◽  
Nikolay Karmanov ◽  
Sergei Kuznetsov
Keyword(s):  
Native Gold ◽  
Microprobe Analysis ◽  
Electron Microprobe Analysis ◽  
Gold Mineralization ◽  
Type I ◽  
Type Ii ◽  
Subpolar Urals ◽  
Type V ◽  
Impurity Elements

Composition of native gold and minerals in intergrowth with rhyolites of the Chudnoe Au-Pd-REE deposit (Subpolar Urals, Russia) was studied using optical microscopy, scanning electron microscopy, and electron microprobe analysis. Five varieties of native gold have been identified, based on the set of impurity elements and their quantities, and on intergrown minerals. Native gold in rhyolites from the Ludnaya ore zone is homogeneous and contains only Ag (fineness 720‰, type I). It is in intergrowth with fuchsite or allanite and mertieite-II. In rhyolites from the Slavnaya ore zone, native gold is heterogeneous, has a higher fineness, different sets and contents of elements: Ag, Cu, 840–860‰ (type II); Ag, Cu, Pd, 830–890‰ (III); Ag, Pd, Cu, Hg, 840–870‰ (IV). It occurs in intergrowth with fuchsite, albite, and mertieite-II (type II), or albite, quartz, and atheneite (III), or quartz, albite, K-feldspar, and mertieite-II (IV). High fineness gold (930–1000‰, type V) with low contents of Ag, Cu, and Pd or their absence occurs in the form as microveins, fringes and microinclusions in native gold II–IV. Tetra-auricupride (AuCu) is presented as isometric inclusions in gold II and platelets in the decay structures in gold III and IV. The preliminary data of a fluid inclusions study showed that gold mineralization at the Chudnoe deposit could have been formed by chloride fluids of low and medium salinity at temperatures from 105 to 230 °C and pressures from 5 to 115 MPa. The formation of native gold I is probably related to fuchsitization and allanitization of rhyolites. The formation of native gold II-V is also associated with the same processes, but it is more complicated and occurred later with a significant role of Na-, Si-, and K-metasomatism. The presence of Pd and Cu in the ores and Cr in fuchsite indicates the important role of mafic-ultramafic magmatism.

scholarly journals Enhanced Cas12a multi-gene regulation using a CRISPR array separator

2021 ◽  
Author(s):  
Jens P. Magnusson ◽  
Antonio R. Rios ◽  
Lingling Wu ◽  
Lei S. Qi
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Gc Content ◽  
Gene Control ◽  
Naturally Occurring ◽  
Crispr Array ◽  
Endogenous Genes ◽  
Simultaneous Activation ◽  
Type V ◽  
Array Performance

AbstractThe type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple crRNAs. Here, we report that Cas12a array performance is hypersensitive to the GC content of crRNA spacers, as high-GC spacers can impair activity of the downstream crRNA. We analyzed naturally occurring CRISPR arrays and observed that repeats always contain an AT-rich fragment that separates crRNAs; we term this fragment a CRISPR separator. Inspired by this observation, we designed short, AT-rich synthetic separators (synSeparators) that successfully removed the disruptive effects between crRNAs. We demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously unknown feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

scholarly journals Distribution of Virulence Genes Among Methicillin-resistant Staphylococcus Aureus of Clinical and Environmental Origin

2020 ◽  
Author(s):  
Deepshikha Bhowmik ◽  
Shiela Chetri ◽  
Bhaskar Jyoti Das ◽  
Debadatta Dhar Chanda ◽  
Amitabha Bhattacharjee
Keyword(s):  
Staphylococcus Aureus ◽  
Multidrug Resistance ◽  
Virulence Genes ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Virulence Gene ◽  
Type I ◽  
Type Ii ◽  
Methicillin Resistant ◽  
Type Iv ◽  
Type V

Abstract Objective: This study was designed to discover the dissemination of virulence genes in Methicillin-resistant Staphylococcus aureus from clinical and environmental settings. Results: The virulence gene such as sea (n=54), seb (n=21), eta (n=27), etb (n=2), cna (n=24), ica (n=2) and tst (n=30) was revealed from this study. Different SCCmec types such as type I, type II, type III, type IV, type V, type VI, type VII, type VIII and type XII were detected among sixty three MRSA isolates where SCCmec type II having ST1551 and type V with ST2416 were found to be associated with multidrug resistance and were highly prevalent in the study area.

scholarly journals Surgical treatment of Neer type II and type V lateral clavicular fractures: comparison of hook plate versus superior plate with lateral extension: a retrospective cohort study

2019 ◽  
Vol 29 (5) ◽  
pp. 989-997 ◽  
Author(s):  
Yassine Ochen ◽  
Herman Frima ◽  
R. Marijn Houwert ◽  
Marilyn Heng ◽  
Mark van Heijl ◽  
...  
Keyword(s):  
Functional Outcome ◽  
Retrospective Cohort ◽  
Removal Rate ◽  
Implant Removal ◽  
Type Ii ◽  
Lateral Extension ◽  
Significant Difference ◽  
Patient Reported ◽  
Outcome Scores ◽  
Type V

Abstract Purpose Different fixation methods are used for treatment of unstable lateral clavicle fractures (LCF). Definitive consensus and guidelines for the surgical fixation of LCF have not been established. The aim of this study was to compare patient-reported functional outcome after open reduction and internal fixation with the clavicle hook plate (CHP) and the superior clavicle plate with lateral extension (SCPLE). Methods A dual-center retrospective cohort study was performed. All patients operatively treated for unstable Neer type II and type V LCF between 2011 and 2016, with the CHP (n = 23) or SCPLE (n = 53), were eligible for inclusion. The primary outcome was the QuickDASH score. Secondary outcomes were the numerical rating scale (NRS) pain score, complications, and implant removal. Results A total of 67 patients (88%) were available for the final follow-up. There was a significant difference in bicortical lateral fragment size, 15 mm (± 4, range 6–21) in the CPH group compared to 20 mm (± 8, range 8–43) in the SCPLE group (p ≤ 0.001). There was no significant difference in median QuickDASH score (CHP; 0.00 [IQR 0.0–0.0], SCPLE; 0.00 [IQR 0.0–4.5]; p = 0.073) or other functional outcome scores (NRS at rest; p = 0.373, NRS during activity; p = 0.559). There was no significant difference in median QuickDASH score or other functional outcome scores between Neer type II and type V fractures. There was no significant difference in complication rate, CHP 11% and SCPLE 8% (relative risk 1.26; [95% CI 0.25–6.33; p = 0.777]). The implant removal rate was 100% in the CHP group compared to 42% in the SCPLE group (relative risk 2.40; [95% CI 1.72–3.35; p ≤ 0.001]). Conclusion Both the CHP and SCPLE are effective fixation methods for the treatment of unstable LCF, resulting in excellent patient-reported functional outcome and similar complication rates. SCPLE fixation is an effective fixation method for the treatment of both Neer type II and type V LCF. The SCPLE has a lower implant removal rate. Therefore, if technically feasible, we recommend SCPLE fixation for the treatment of unstable LCF.

Syntheses of Post-Iboga Alkaloids

Synthesis ◽  
2019 ◽  
Vol 51 (14) ◽  
pp. 2737-2758 ◽  
Author(s):  
Hyeonggeun Lim ◽  
Sikwang Seong ◽  
Sunkyu Han
Keyword(s):  
Secondary Metabolites ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Type Iv ◽  
Synthetic Studies ◽  
Type V

Post-iboga alkaloids are secondary metabolites that are biosynthetically derived from iboga-type alkaloids via rearrangements of the indole and/or isoquinuclidine moieties. Herein, we categorize post-iboga alkaloids into five types based on the biosynthetic mode of transformation of the iboga scaffold. We then describe reported syntheses of post-iboga alkaloids, including our laboratory’s recent contributions, based on our own categorization.1 Introduction1.1 Iboga and Post-Iboga Alkaloids1.2 Classification of Post-Iboga Alkaloids1.2.1 Introduction to Type I Post-Iboga Alkaloids1.2.2 Introduction to Type II Post-Iboga Alkaloids1.2.3 Introduction to Type III Post-Iboga Alkaloids1.2.4 Introduction to Type IV Post-Iboga Alkaloids1.2.5 Introduction to Type V Post-Iboga Alkaloids2 Syntheses of Post-Iboga Alkaloids2.1 Syntheses of Type I Post-Iboga Alkaloids2.1.1 Syntheses of Monomeric Type I Post-Iboga Alkaloids2.1.2 Syntheses of Dimeric Type I Post-Iboga Alkaloids2.2 Syntheses of Type II Post-Iboga Alkaloids2.3 Synthetic Studies Toward Type III Post-Iboga Alkaloids2.4 Syntheses of Type IV Post-Iboga Alkaloids2.5 Synthesis of Type V Post-Iboga Alkaloids3 Conclusion and Outlook

scholarly journals Biomechanical Impact of FHL Tendon Harvest on Forefoot and Great Toe Push-off Strength and Its Correlation to Knot of Henry Crossover Variation

2018 ◽  
Vol 3 (3) ◽  
pp. 2473011418S0003
Author(s):  
Timothy Charlton ◽  
Danielle Thomas ◽  
David Thordarson ◽  
Melodie Metzger ◽  
Trevor Nelson
Keyword(s):  
Repeated Measures ◽  
Plantar Flexion ◽  
Tissue Bank ◽  
Flexor Hallucis Longus ◽  
Type I ◽  
Type Ii ◽  
Great Toe ◽  
Type Iv ◽  
Loading Patterns ◽  
Type V

Category: Midfoot/Forefoot Introduction/Purpose: The flexor hallucis longus (FHL) tendon is commonly used for tendon transfers in reconstructive Achilles tendon procedures. A subset of patients who undergo this procedure complain of first great toe weakness and loss of push off strength after FHL tendon transfer. Despite the frequency of this procedure, there is currently little information available to surgeons to help understand this potential complication. Therefore, the objective of this biomechanical cadaveric study was to quantify plantar flexion strength after FHL harvest and correlate it to variations in anatomy to determine if distinct tendon crossover patterns at the Knot of Henry are more likely to lead to forefoot weakness. Methods: Cadaveric specimens were procured from an approved tissue bank. The proximal end of the tibia was potted and secured to the Mechanical Testing System. A pressure mapping system was used to measure plantar force though the great toe and lesser toes. The Achilles, FHL, and flexor digitorum longus (FDL) tendons were attached to linear actuators for load application. Pressure under the toes was measured with the Achilles alone, Achilles with FHL, Achilles with FDL, and Achilles with both FHL and FDL. The resultant loading patterns were recorded in the greater and lesser toes and compared between the different states. After biomechanical testing, all specimens were carefully dissected and the tendinous slips between the FHL and FDL were documented and classified based on a previously determined system (Types I-V, LaRue; Edama) Functional and anatomical relationship between the classification type and loading patterns were statistically analyzed using repeated measures ANOVA. Results: 23 specimens (13M / 10F) with a mean age of 71 years (range: 54-90) were used in this study. 61% were anatomically classified as type I, followed by type II (26%), IV (9%), and type V (4%), and type III (0%). Simulated FHL harvest in type I specimens resulted in a 29% decrease in great toe flexion pressure and an 21% decrease in total forefoot flexion pressure, p<0.05. In type II feet, FHL harvest led to a greater reduction in flexion pressure in the great toe (34%) and forefoot (25%), p<0.05. Type IV specimens also had a decrease in flexion pressure in both the great toe (21%) and forefoot (15%), p<0.05. Type V specimens trended similar to type I specimens. Conclusion: This study is the first to quantify loss of great toe and lesser toe flexion pressure after FHL harvest. In addition, it is the first to correlate these losses to variations in anatomic crossover patterns at the Knot of Henry. Specimens classified as type II had the greatest reduction in flexion pressure, followed by type I and type IV. This information is clinically important for preoperative discussions about post-surgical expectations and surgical planning.

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