Design of time-delayed safety switches for CRISPR gene therapy.
Ontology highlight
ABSTRACT:
SUBMITTER: Sun D
PROVIDER: S-EPMC8377138 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The potential of adoptive T-cell therapy in effecting complete and durable responses has been demonstrated in a number of malignant and infectious diseases. Ongoing progress in T-cell engineering has given cause for optimism in the broader clinical applicability of this approach. However, the development of more potent T cells is checked by safety concerns, highlighted by the occurrence of on-target and off-target toxicities that, although uncommon, have been fatal on occasions. Timely pharmacological intervention is effective in the management of a majority of adverse events but adoptively transferred T cells can persist long term, along with any unwanted effects. A recently validated cellular safety switch, inducible caspase 9 (iCasp9), has the potential to mitigate the risks of T-cell therapy by enabling the elimination of transferred T cells if required. In haematopoietic stem cell transplantation, iCasp9-modified donor T cells can be rapidly eliminated in the event of graft-versus-host disease. This review presents an overview of the risks associated with modern T-cell therapy and the development, clinical results and potential future application of the iCasp9 safety switch.
| S-EPMC4232067 | biostudies-literature
Project description:CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT.
| S-EPMC5757841 | biostudies-literature
Project description:The main (Mpro) and papain-like (PLpro) proteases encoded by SARS-CoV-2 are essential to process viral polyproteins into functional units, thus representing key targets for anti-viral drug development. There is a need for an efficient inhibitor screening system that can identify drug candidates in a cellular context. Here we describe modular, tunable autoproteolytic gene switches (TAGS) relying on synthetic transcription factors that self-inactivate, unless in the presence of coronavirus protease inhibitors, consequently activating transgene expression. TAGS rapidly report the impact of drug candidates on Mpro and PLpro activities with a high signal-to-noise response and a sensitivity matching concentration ranges inhibiting viral replication. The modularity of the TAGS enabled the study of other Coronaviridae proteases, characterization of mutations and multiplexing of gene switches in human cells. Mice implanted with Mpro or PLpro TAGS-engineered cells enabled analysis of the activity and bioavailability of protease inhibitors in vivo in a virus-free setting.
| S-EPMC8609006 | biostudies-literature
Project description:Control of biological populations remains a critical goal to address the challenges facing ecosystems and agriculture and those posed by human disease, including pests, parasites, pathogens and invasive species. A particular architecture of the CRISPR/Cas biotechnology - a gene drive - has the potential to modify or eliminate populations on a massive scale. Super-Mendelian inheritance has now been demonstrated in both fungi and metazoans, including disease vectors such as mosquitoes. Studies in yeast and fly model systems have developed a number of molecular safeguards to increase biosafety and control over drive systems in vivo, including titration of nuclease activity, anti-CRISPR-dependent inhibition and use of non-native DNA target sites. We have developed a CRISPR/Cas9 gene drive in Saccharomyces cerevisiae that allows for the safe and rapid examination of alternative drive designs and control mechanisms. In this study, we tested whether non-homologous end-joining (NHEJ) had occurred within diploid cells displaying a loss of the target allele following drive activation and did not detect any instances of NHEJ within multiple sampled populations. We also demonstrated successful multiplexing using two additional non-native target sequences. Furthermore, we extended our analysis of 'resistant' clones that still harboured both the drive and target selection markers following expression of Streptococcus pyogenes Cas9; de novo mutation or NHEJ-based repair could not explain the majority of these heterozygous clones. Finally, we developed a second-generation gene drive in yeast with a guide RNA cassette integrated within the drive locus with a near 100 % success rate; resistant clones in this system could also be reactivated during a second round of Cas9 induction.
| S-EPMC7472540 | biostudies-literature
Project description:UnlabelledThe CRISPR/Cas9 system was recently developed as a powerful and flexible technology for targeted genome engineering, including genome editing (altering the genetic sequence) and gene regulation (without altering the genetic sequence). These applications require the design of single guide RNAs (sgRNAs) that are efficient and specific. However, this remains challenging, as it requires the consideration of many criteria. Several sgRNA design tools have been developed for gene editing, but currently there is no tool for the design of sgRNAs for gene regulation. With accumulating experimental data on the use of CRISPR/Cas9 for gene editing and regulation, we implement a comprehensive computational tool based on a set of sgRNA design rules summarized from these published reports. We report a genome-wide sgRNA design tool and provide an online website for predicting sgRNAs that are efficient and specific. We name the tool CRISPR-ERA, for clustered regularly interspaced short palindromic repeat-mediated editing, repression, and activation (ERA).Availability and implementationhttp://CRISPR-ERA.stanford.edu.Contactstanley.qi@stanford.edu or xwwang@tsinghua.edu.cnSupplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC4757951 | biostudies-literature
Project description:BackgroundIt is one of the ultimate goals for modern biological research to fully elucidate the intricate interplays and the regulations of the molecular determinants that propel and characterize the progression of versatile life phenomena, to name a few, cell cycling, developmental biology, aging, and the progressive and recurrent pathogenesis of complex diseases. The vast amount of large-scale and genome-wide time-resolved data is becoming increasing available, which provides the golden opportunity to unravel the challenging reverse-engineering problem of time-delayed gene regulatory networks.ResultsIn particular, this methodological paper aims to reconstruct regulatory networks from temporal gene expression data by using delayed correlations between genes, i.e., pairwise overlaps of expression levels shifted in time relative each other. We have thus developed a novel model-free computational toolbox termed TdGRN (Time-delayed Gene Regulatory Network) to address the underlying regulations of genes that can span any unit(s) of time intervals. This bioinformatics toolbox has provided a unified approach to uncovering time trends of gene regulations through decision analysis of the newly designed time-delayed gene expression matrix. We have applied the proposed method to yeast cell cycling and human HeLa cell cycling and have discovered most of the underlying time-delayed regulations that are supported by multiple lines of experimental evidence and that are remarkably consistent with the current knowledge on phase characteristics for the cell cyclings.ConclusionWe established a usable and powerful model-free approach to dissecting high-order dynamic trends of gene-gene interactions. We have carefully validated the proposed algorithm by applying it to two publicly available cell cycling datasets. In addition to uncovering the time trends of gene regulations for cell cycling, this unified approach can also be used to study the complex gene regulations related to the development, aging and progressive pathogenesis of a complex disease where potential dependences between different experiment units might occurs.
| S-EPMC1386718 | biostudies-literature
Project description:Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering.
| S-EPMC6733528 | biostudies-literature
Project description:Protein conformational switches alter their shape upon receiving an input signal, such as ligand binding, chemical modification, or change in environment. The apparent simplicity of this transformation--which can be carried out by a molecule as small as a thousand atoms or so--belies its critical importance to the life of the cell as well as its capacity for engineering by humans. In the realm of molecular switches, proteins are unique because they are capable of performing a variety of biological functions. Switchable proteins are therefore of high interest to the fields of biology, biotechnology, and medicine. These molecules are beginning to be exploited as the core machinery behind a new generation of biosensors, functionally regulated enzymes, and "smart" biomaterials that react to their surroundings. As inspirations for these designs, researchers continue to analyze existing examples of allosteric proteins. Recent years have also witnessed the development of new methodologies for introducing conformational change into proteins that previously had none. Herein we review examples of both natural and engineered protein switches in the context of four basic modes of conformational change: rigid-body domain movement, limited structural rearrangement, global fold switching, and folding-unfolding. Our purpose is to highlight examples that can potentially serve as platforms for the design of custom switches. Accordingly, we focus on inducible conformational changes that are substantial enough to produce a functional response (e.g., in a second protein to which it is fused), yet are relatively simple, structurally well-characterized, and amenable to protein engineering efforts.
| S-EPMC3404493 | biostudies-literature
Project description:E.coli K-12 W3110 was grown in LB medium and harvested at each time point. And time-series microarray experiments were performed based on Sequential Design. In Sequential Design, the control sample is set to closest previous time point so that adjacent time points are compared directly. Combining with data from reference design, more accurate and reliable expression series could be collected. Keywords: timecourse
2018-03-01 | GSE6100 | GEO
Project description:E.coli K-12 W3110 was grown in LB medium and harvested at each time point. And time series microarray experiments were performed based on reference desgin. In reference design, the control sample is collected at one representative time point. Combining with data from sequential design, more acculate and reliable expression series could be collected. Keywords: Reference design
2011-03-31 | GSE6033 | GEO