gene-digital-circuits-based-on-crispr-cas-systems-anti-crispr

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

in-vitro-preparation-actively-maturing-bovine-articular-cartilage

In Vitro Preparation of Actively Maturing Bovine Articular Cartilage Explants for X-Ray Phase Contrast Imaging

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of ...

Swansea University Medical School

Tianjin University

Platelet-rich plasma (PRP) is used to stimulate the repair of acute and chronic cartilage damage even though there is no definitive evidence of how this is achieved. Chondrocytes in injured and diseased situations frequently re-express phenotypic biomarkers of immature cartilage so tissue maturation is a potential pathway for restoration of normal structure and function. We used an in vitro model of growth factor-induced maturation to perform a comparative study in order to determine whether PRP can also induce this specific form of remodeling that is characterised by increased cellular proliferation and tissue stiffness. Gene expression patterns specific for maturation were mimicked in PRP treated cartilage, with chondromodulin, collagen types II/X downregulated, deiodinase II and netrin-1 upregulated. PRP increased cartilage surface cell density 1.5-fold (P < 0.05), confirmed by bromodeoxyuridine incorporation and proportionate increases in proliferating cell nuclear antigen gene expression. Atomic force microscopy analysis of PRP and growth factor treated cartilage gave a 5-fold increase in stiffness correlating with a 10-fold upregulation of lysyl oxidase like-1 gene expression (P < 0.001). These data show PRP induces key aspects of post-natal maturation in immature cartilage and provides the basis to evaluate a new biological rationale for its activity when used clinically to initiate joint repair.

Platelet-rich plasma induces post-natal maturation of immature articular cartilage and correlates with LOXL1 activation.

Cell-laden hydrogels are the primary building blocks for bioprinting, and, also termed bioinks, are the foundations for creating structures that can potentially recapitulate the architecture of articular cartilage. To be functional, hydrogel constructs need to unlock the regenerative capacity of encapsulated cells. The recent identification of multipotent articular cartilage-resident chondroprogenitor cells (ACPCs), which share important traits with adult stem cells, represents a new opportunity for cartilage regeneration. However, little is known about the suitability of ACPCs for tissue engineering, especially in combination with biomaterials. This study aimed to investigate the potential of ACPCs in hydrogels for cartilage regeneration and biofabrication, and to evaluate their ability for zone-specific matrix production. Gelatin methacryloyl (gelMA)-based hydrogels were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes, and as bioinks for printing. Our data shows ACPCs outperformed chondrocytes in terms of neo-cartilage production and unlike MSCs, ACPCs had the lowest gene expression levels of hypertrophy marker collagen type X, and the highest expression of PRG4, a key factor in joint lubrication. Co-cultures of the cell types in multi-compartment hydrogels allowed generating constructs with a layered distribution of collagens and glycosaminoglycans. By combining ACPC- and MSC-laden bioinks, a bioprinted model of articular cartilage was generated, consisting of defined superficial and deep regions, each with distinct cellular and extracellular matrix composition. Taken together, these results provide important information for the use of ACPC-laden hydrogels in regenerative medicine, and pave the way to the biofabrication of 3D constructs with multiple cell types for cartilage regeneration or in vitro tissue models.

The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells.

Nasoseptal cartilage has been assumed to be isotropic, unlike the well-defined zonal organization of articular cartilage attributed to postnatal biomechanical loading. We know from clinical experience that malrotation of surgical nasoseptal cartilage grafts can lead to increased graft absorption. Other studies have also suggested directionally dependent compressive stiffness suggesting anisotropy, but morphological investigations are lacking. This study characterizes immature and mature native bovine nasoseptal cartilage using a combination of immunohistochemistry, biomechanical testing and structural imaging. Our findings indicate that there is extensive postnatal synthesis and reorganization of the extracellular matrix in bovine nasoseptal cartilage, independent of joint loading forces responsible for articular cartilage anisotropy. Immature nasoseptal cartilage is more cellular and homogenous compared to the zonal organization of cells and extracellular matrix of mature cartilage. Mature samples also exhibited greater glycosaminoglycan content and type II collagen fibre alignment compared to immature cartilage and this correlates with greater compressive stiffness. Engineered neocartilage often consists of immature, isotropic, homogenous tissue that is unable to meet the functional and mechanical demands when implanted into the native environment. This study demonstrates the importance of anisotropy on biomechanical tissue strength to guide future cartilage tissue engineering strategies for surgical reconstruction.

Morphological and biomechanical characterization of immature and mature nasoseptal cartilage.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Morphological and biomechanical characterization of immature and mature nasoseptal cartilage.

Articular cartilage contains a subpopulation of tissue-specific progenitors that are an ideal cell type for cell therapies and generating neocartilage for tissue engineering applications. However, it is unclear whether the standard chondrogenic medium using transforming growth factor beta (TGFβ) isoforms is optimal to differentiate these cells. We therefore used pellet culture to screen progenitors from immature bovine articular cartilage with a number of chondrogenic factors and discovered that bone morphogenetic protein-9 (BMP9) precociously induces their differentiation. This difference was apparent with toluidine blue staining and confirmed by biochemical and transcriptional analyses with BMP9-treated progenitors exhibiting 11-fold and 5-fold greater aggrecan and collagen type II (COL2A1) gene expression than TGFβ1-treated progenitors. Quantitative gene expression analysis over 14 days highlighted the rapid and phased nature of BMP9-induced chondrogenesis with sequential activation of aggrecan then collagen type II, and negligible collagen type X gene expression. The extracellular matrix of TGFβ1-treated progenitors analyzed using atomic force microscopy was fibrillar and stiff whist BMP9-induced matrix of cells more compliant and correspondingly less fibrillar. Polarized light microscopy revealed an annular pattern of collagen fibril deposition typified by TGFβ1-treated pellets, whereas BMP9-treated pellets displayed a birefringence pattern that was more anisotropic. Remarkably, differentiated immature chondrocytes incubated as high-density cultures in vitro with BMP9 generated a pronounced anisotropic organization of collagen fibrils indistinguishable from mature adult articular cartilage, with cells in deeper zones arranged in columnar manner. This contrasted with cells grown with TGFβ1, where a concentric pattern of collagen fibrils was visualized within tissue pellets. In summary, BMP9 is a potent chondrogenic factor for articular cartilage progenitors and is also capable of inducing morphogenesis of adult-like cartilage, a highly desirable attribute for in vitro tissue-engineered cartilage.

Bone Morphogenetic Protein-9 Is a Potent Chondrogenic and Morphogenic Factor for Articular Cartilage Chondroprogenitors.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) system represents, in prokaryotes, an adaptive and inheritable immune response against invading DNA. The discovery of anti-CRISPR proteins (Acrs), which are inhibitors of CRISPR-Cas, mainly encoded by phages and prophages, showed a co-evolution history between prokaryotes and phages. In the past decade, the CRISPR-Cas systems together with the corresponding Acrs have been turned into a genetic-engineering tool. Among the six types of CRISPR-Cas characterized so far, type II CRISPR-Cas system is the most popular in biotechnology. Here, we discuss about the discovery, the reported inhibitory mechanisms, and the applications in both gene editing and gene transcriptional regulation of type II Acrs. Moreover, we provide insights into future potential research and feasible applications.

Type II anti-CRISPR proteins as a new tool for synthetic biology.

Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation.

Type II CRISPR-(d)SpCas9 and anti-CRISPR proteins (AcrIIs) show evidence of coevolution and competition for survival between bacteria and phages. In biotechnology, CRISPR-(d)SpCas9 is utilized for gene editing and transcriptional regulation. Moreover, its activity is controlled by AcrIIs. However, studies of dSpCas9/AcrII-based transcription regulation in  are rare. In this work, we used dSpCas9 as a template to engineer new transcription activators. We found that the most performant activation system requires the use of bare dSpCas9 in conjunction with scaffold gRNA (scRNA). This means that activation domains shall not be fused to dSpCas9 but rather interact with scRNA. We showed that a low amount of sgRNA is not a limiting factor in dSpCas9-driven transcription regulation. Moreover, a high quantity of sgRNA does not improve, generally, activation (and repression) efficiency. Importantly, we analyzed the performance of AcrIIA2, AcrIIA4, and AcrIIA5 in  in depth. AcrIIA4 is the strongest of the three AcrIIs and also the only one able to induce high inhibition at low concentrations. However, the activation domains fused to dSpCas9 hindered interactions with the AcrIIs as well and limited their control of gene transcription regulation, confirming that bare dSpCas9 is the best solution for building synthetic genetic networks in yeast.

Interaction of Bare dSpCas9, Scaffold gRNA, and Type II Anti-CRISPR Proteins Highly Favors the Control of Gene Expression in the Yeast .

CRISPR-Cas systems provide powerful biological tools for genetic manipulation and gene expression regulation. Class 2 systems, comprising type II, type V, and type VI, have the significant advantage to require a single effector Cas protein (Cas9, Cas12, and Cas13 respectively) to cleave nucleic acids upon binding the crRNA. Both Cas9 and Cas12 recognize DNA and induce a double-strand break in it. In contrast, Cas13 bind and cleave RNA exclusively. However, some Cas9 homologs have shown RNase activity as well. Here, we harnessed Nme1Cas9, LwaCas13a, and RfxCas13d to carry out gene downregulation in  by triggering mRNA degradation. To avoid potential DNA damage, we mutated Nme1Cas9 into dNme1Cas9 that lost the nuclease activity of the RuvC domain but retained the active HNH domain, able to act on the target DNA strand and, therefore, on the corresponding transcript. Our results showed that dNme1Cas9 is a functional RNase  although with moderate activity since it provoked a fluorescence reduction from 21% to 32%. Interestingly, dNme1Cas9 works in a PAM-independent way nor demands helper PAMmer molecules. LwaCas13a and RfxCas13d appeared substantially unfunctional in , though they were shown to perform well in mammalian cells. To the best of our knowledge, this is the first report about the working  of a variant of Nme1Cas9 as an RNase and the issues connected with the usage of Cas13 proteins in .

ArticleTotal : 2	Year
Publication title Cited by 6	2022
Publication title	2022

Article	Year
Platelet-rich plasma induces post-natal maturation of immature articular cartilage and correlates with LOXL1 activation. Scientific reports\| PubMed ID: 28623328	2017
The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells. Acta biomaterialia\| PubMed ID: 28782725	2017
Morphological and biomechanical characterization of immature and mature nasoseptal cartilage. Scientific reports\| PubMed ID: 31462660	2019
Author Correction: Morphological and biomechanical characterization of immature and mature nasoseptal cartilage. Scientific reports\| PubMed ID: 32157206	2020
Bone Morphogenetic Protein-9 Is a Potent Chondrogenic and Morphogenic Factor for Articular Cartilage Chondroprogenitors. Stem cells and development\| PubMed ID: 32364057	2020
Type II anti-CRISPR proteins as a new tool for synthetic biology. RNA biology\| PubMed ID: 32991234	2021
Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation. Circulation research\| PubMed ID: 34111951	2021
Interaction of Bare dSpCas9, Scaffold gRNA, and Type II Anti-CRISPR Proteins Highly Favors the Control of Gene Expression in the Yeast . ACS synthetic biology\| PubMed ID: 34994540	2022
A Mutated Nme1Cas9 Is a Functional Alternative RNase to Both LwaCas13a and RfxCas13d in the Yeast . Frontiers in bioengineering and biotechnology\| PubMed ID: 35721864	2022

Yadan Zhang

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