Introduction Of Insertion Research Articles

Tobacco packaging littering behaviours among Australian adults who smoke: findings from cross-sectional and observational studies to inform the implementation of cigarette pack inserts

BackgroundCigarette pack inserts are small cards that highlight the benefits of quitting and promote use of smoking cessation support. With evidence from Canada that they increase self-efficacy to quit, quit...

Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

New technologies and large-cohort studies have enabled novel variant discovery and association at unprecedented scale, yet functional characterization of these variants remains paramount to deciphering disease mechanisms. Approaches that facilitate parallelized genome editing of cells of interest or induced pluripotent stem cells (iPSCs) have become critical tools toward this goal. Here, we developed an approach that incorporates libraries of CRISPR-Cas9 guide RNAs (gRNAs) together with inducible Cas9 into a piggyBac (PB) transposon system to engineer dozens to hundreds of genomic variants in parallel against isogenic cellular backgrounds. This method empowers loss-of-function (LoF) studies through the introduction of insertions or deletions (indels) and copy-number variants (CNVs), though generating specific nucleotide changes is possible with prime editing. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of isogenic cellular collections and catalyze comparative functional genomic studies investigating the roles of hundreds of genes and mutations in development and disease.

In Vivo Delivery of RNA By Lipid Nanoparticles (LNP) to Hematopoietic Stem Cells and T Cells in Humanized Mice and Non-Human Primates

Genomic medicines have significant therapeutic potential to cure rare genetic diseases and cancer, but currently, most treatments rely on ex vivo cell editing, which is time consuming, costly, and suffers from logistic and manufacturing challenges. In contrast, in vivo delivery of genomic medicines would unlock new therapeutic areas and provide broader patient access. However, the success of in vivo delivery is dependent on the development of safe delivery vehicles capable of transporting the genomic medicines to their intended target tissues and cells. In recent years, lipid nanoparticles (LNPs) have emerged as versatile delivery solutions for RNA therapeutics but have seldom been used fo r in vivo delivery beyond liver and muscle tissue. Challenges in extrahepatic delivery of LNPs include adequate tissue-specific targeting to achieve therapeutic levels of genome editing while avoiding anti-drug immune responses. We have developed novel LNPs designed to deliver RNA payloads to either T cells or hematopoietic stem cells (HSCs) in vivo. Using our novel T-cell LNPs, we have demonstrated single-dose in vivo delivery of GFP mRNA in two humanized mouse models. In the spleen of humanized NSG mice (pre-engrafted with human HSCs 12-weeks prior to study), we achieved GFP expression in 24% of human CD3 + T cells. In another model, NSG mice infused with human T cells 14-days prior to study, GFP expression was observed in 76% of T cells in the spleen. Single-dose administration of this T-cell LNP in rhesus macaque resulted in 40% GFP expression in peripheral blood T cells. Furthermore, we did not observe any detectable GFP expression in human or rhesus B cells, suggesting this LNP system may be suitable for the development of in vivo CAR-T therapies for B cell malignancies. Separately, we treated humanized NBSGW mice (pre-engrafted with human HSCs for 12 weeks) with a single dose of our HSC LNPs delivering GFP mRNA. Expression of GFP was observed in approximately 90% of bone marrow hematopoietic stem and progenitor cells (HSPCs defined as Lin -CD34 +CD38 - cells) and approximately 95% of long-term bone marrow HSC-enriched population (HSCs defined as Lin -CD34 +CD38 -CD90 +CD45RA - cells). Single-dose intravenous administration of the same LNP in cynomolgus macaques led to GFP expression in 61% of HSPCs and 62% of HSCs in the bone marrow. Gene editing presents a greater challenge than reporter mRNA delivery due to the increased size and complexity of the RNA cargos. Toward this goal, we went on to demonstrate that our HSC LNP platform can deliver Cas9 mRNA, with a beta-2 microglobulin (B2M) sgRNA, to human HSCs in humanized NBSGW mice resulting in the knockout (KO) of the B2M gene through the introduction of insertion or deletion mutations. Likewise, following administration of a B2M editing T-cell LNP in humanized NSG mice, we demonstrated B2M KO in splenic human CD3 + T cells. Together, these in vivo delivery results indicate our LNP delivery platform is capable of efficiently delivering RNA payloads to different hematopoietic cell types in blood and tissues, providing a potential delivery platform for in vivo genome editing.

Detection of unintended on-target effects in CRISPR genome editing by DNA donors carrying diagnostic substitutions.

CRISPR nucleases can introduce double-stranded DNA breaks in genomes at positions specified by guide RNAs. When repaired by the cell, this may result in the introduction of insertions and deletions or nucleotide substitutions provided by exogenous DNA donors. However, cellular repair can also result in unintended on-target effects, primarily larger deletions and loss of heterozygosity due to gene conversion. Here we present a strategy that allows easy and reliable detection of unintended on-target effects as well as the generation of control cells that carry wild-type alleles but have demonstratively undergone genome editing at the target site. Our 'sequence-ascertained favorable editing' (SAFE) donor approach relies on the use of DNA donor mixtures containing the desired nucleotide substitutions or the wild-type alleles together with combinations of additional 'diagnostic' substitutions unlikely to have any effects. Sequencing of the target sites then results in that two different sequences are seen when both chromosomes are edited with 'SAFE' donors containing different sets of substitutions, while a single sequence indicates unintended effects such as deletions or gene conversion. We analyzed more than 850 human embryonic stem cell clones edited with 'SAFE' donors and detect all copy number changes and almost all clones with gene conversion.

Delivery Aspects of CRISPR/Cas for in Vivo Genome Editing.

ConspectusThe discovery of CRISPR/Cas has revolutionized the field of genome editing. CRIPSR/Cas components are part of the bacterial immune system and are able to induce double-strand DNA breaks in the genome, which are resolved by endogenous DNA repair mechanisms. The most relevant of these are the error-prone nonhomologous end joining and homology directed repair pathways. The former can lead to gene knockout by introduction of insertions and deletions at the cut site, while the latter can be used for gene correction based on a provided repair template. In this Account, we focus on the delivery aspects of CRISPR/Cas for therapeutic applications in vivo. Safe and effective delivery of the CRISPR/Cas components into the nucleus of affected cells is essential for therapeutic gene editing. These components can be delivered in several formats, such as pDNA, viral vectors, or ribonuclear complexes. In the ideal case, the delivery system should address the current limitations of CRISPR gene editing, which are (1) lack of targeting specific tissues or cells, (2) the inability to enter cells, (3) activation of the immune system, and (4) off-target events.To circumvent most of these problems, initial therapeutic applications of CRISPR/Cas were performed on cells ex vivo via classical methods (e.g., microinjection or electroporation) and novel methods (e.g., TRIAMF and iTOP). Ideal candidates for such methods are, for example, hematopoietic cells, but not all tissue types are suited for ex vivo manipulation. For direct in vivo application, however, delivery systems are needed that can target the CRISPR/Cas components to specific tissues or cells in the human body, without causing immune activation or causing high frequencies of off-target effects.Viral systems have been used as a first resort to transduce cells in vivo. These systems suffer from problems related to packaging constraints, immunogenicity, and longevity of Cas expression, which favors off-target events. Viral vectors are as such not the best choice for direct in vivo delivery of CRISPR/Cas. Synthetic vectors can deliver nucleic acids as well, without the innate disadvantages of viral vectors. They can be classed into lipid, polymeric, and inorganic particles, all of which have been reported in the literature. The advantage of synthetic systems is that they can deliver the CRISPR/Cas system also as a preformed ribonucleoprotein complex. The transient nature of this approach favors low frequencies of off-target events and minimizes the window of immune activation. Moreover, from a pharmaceutical perspective, synthetic delivery systems are much easier to scale up for clinical use compared to viral vectors and can be chemically functionalized with ligands to obtain target cell specificity. The first preclinical results with lipid nanoparticles delivering CRISPR/Cas either as mRNA or ribonucleoproteins are very promising. The goal is translating these CRISPR/Cas therapeutics to a clinical setting as well. Taken together, these current trends seem to favor the use of sgRNA/Cas ribonucleoprotein complexes delivered in vivo by synthetic particles.

Characteristics of Genome Editing Mutations in Cereal Crops

Designer nucleases allow the creation of new plant genotypes by introducing precisely-targeted double-strand breaks that are resolved by endogenous repair pathways. The major nuclease technologies are meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and the CRISPR/Cas9 system. Each comprises a promiscuous endonuclease guided by protein-DNA or RNA-DNA interactions. A great deal is known about the principles of designer nucleases but much remains to be learned about their detailed behavioral characteristics in different plant species. The outcome of genome engineering reflects the intrinsic properties of each nuclease and target genome, causing variations in efficiency, accuracy, and mutation structure. In this article, we critically discuss the activities of designer nucleases in different cereals representing a broad range of genome characteristics.

CRISPR/Cas9: a promising way to exploitgenetic variation in plants.

Creation of variation in existing gene pool of crop plants is the foremost requirement in crop improvement programmes. Genome editing is a tool to produce knock out of target genes either by introduction of insertion or by deletion that disrupts the function of a specific gene. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system is the most recent addition to the toolbox of sequence-specific nucleases that includes ZFNs and TALENs. The CRISPR/Cas9 system allows targeted cleavage of genomic DNA guided by a small noncoding RNA, resulting in gene modifications by both non-homologous end joining and homology-directed repair mechanisms. Here, we present an overview of mechanisms of CRISPR, its potential roles in creating variation in germplasm and applications of this novel interference pathway in crop improvement. The availability of the CRISPR/Cas9 system holds promise in facilitating both forward and reverse genetics and will enhance research in crops that lack genetic resources.

Autophagy Controls Exosomal Release of Prions and Lateral Prion Infection

Prion diseases are fatal transmissible neurodegenerative disorders that affect both human and animals. They are characterized by conformational conversion of the normal cellular prion protein (PrPc) into a disease associated pathological isoform (PrPSc). It has been shown that PrPSc can infect recipient cells when it is packaged into endosomal derived nanoparticles called exosomes. Exosomes are present in all biological fluids such as blood, urine, milk, and saliva. Exosome secretion is under the influence of the autophagic system, a basic cellular degradation machinery. Autophagy stimulation can inhibit exosomal release, whereas inhibition of autophagy seems to enhance exosomal release. In this paradigm, our work investigates the effect of autophagy modulation on exosomal release of prions and how this interplay impacts prion infection. We successfully isolated and characterized exosomes from a prion‐infected neuronal cell line (ScN2a) using differential ultracentrifugation technique. The isolated exosomes contained PrPSc as shown by immunoblot. Treatment of ScN2a cells with the neutral sphingomyelinase inhibitor GW4869 resulted in a block of the exosomal release and reduced PrPSc levels in exosomes. When we stimulated autophagy in ScN2a cells using rapamycin, a well‐known autophagy stimulator that inhibits the mammalian target of Rapamycin (mTOR) pathway, we observed a strong inhibition of exosomal release and decreased levels of PrPSc in exosomes compared to vehicle treated cells. To further assess the impact of autophagy on exosomal prion release, we knocked‐out Atg5 (a main player in the autophagic machinery) in N2a cells using the CRISPR‐Cas9 system. CRISPR‐Cas9 is the clustered, regularly interspaced, short palindromic repeats (CRISPR)‐associated protein (Cas) system which involves RNA–guided site specific DNA double strand cleavages. Non‐homologous end joining is used by cells for repair of double strand breaks and may result in functional gene knock‐out by introduction of insertions and deletions. Using this technology we generated various N2a cell clones with functional knockouts in different exons of the Atg5 gene, as verified by sequencing and immunoblot analysis. Upon stably infecting these Atg5 knock‐out cells with prions, we found a highly increased release of exosomes and exosome‐associated PrPSc compared to wild type cells. Taken together, our data shows that autophagy modulation can control lateral prion infection by interfering with exosomal release of PrPSc. Our present work correlates these findings to outcomes of prion infection in recipient cells and in mouse animal models. Overall, our study describes a novel interplay of basic cellular machinery which affects the live cycle of prions. This new understanding will result in novel targets for therapy against prion diseases and protein misfolding disorders.Support or Funding InformationThe Natural Sciences and Engineering Research Council of Canada (NSERC) Alberta Prion Research Institute (APRI) National Institute of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) Alberta Innovates Health Solutions (AIHS)

Routine Markerless Gene Replacement in Bacillus anthracis

An improved genetic tool suitable for routine markerless allelic exchange in Bacillus anthracis has been constructed. Its utility was demonstrated by the introduction of insertions, deletions, and missense mutations on the chromosome and plasmid pXO1 of the Sterne strain of B. anthracis.

Ceramic Cross-Flow Microfiltration in the Presence of Inserts

The influence of inserts with various configurations on permeate flux through a tubular ceramic microfiltration membrane was studied. The commercial metal-working oil emulsion was used in the experiments. A more extensive study of the effect of shear rate on permeate flux was made by comparing the steady-state flux of the empty tube system and the system with inserts. The cross-flow microfiltration with inserts was found to be simple and effective. Analysing experimental results, it was concluded that the introduction of inserts, irrespective of their configurations, resulted in a significant increase in permeate flux compared with results obtained in the empty tube system. The interruption of formation of a boundary layer by the inserts is attributed to mixing and migration of rejected particles from the membrane surface.

An investigation on the configuration of inserts in tubular ultrafiltration module to control concentration polarization

The flux through a tubular ultrafiltration membrane has been improved using inserts with different configurations in the module. It is established that the introduction of inserts, irrespective of their configuration, leads to better flux in comparison with that in the empty tube. Inserts with variable cross-sectional area show better performances than do smooth cylindrical ones. The interruption of the formation of a gel layer by the inserts with variable cross-sectional area is attributed to the periodical change in the feed flow velocity and the tangential stress on the membrane surface.