Search results for "CRISPR"

The application of the CRISPR-Cas9 genome editing machinery in food and agricultural science: Current status, future perspectives, and associated cha…

2019

The recent progress in genetic engineering has brought multiple benefits to the food and agricultural industry by enhancing the essential characteristics of agronomic traits. Powerful tools in the field of genome editing, such as siRNA-mediated RNA interference for targeted suppression of gene expression and transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) for DNA repair have been widely used for commercial purposes. However, in the last few years, the discovery of the CRISPR-Cas9 system has revolutionized genome editing and has attracted attention as a powerful tool for several industrial applications. Herein, we review current progresses in the uti…

CRISPR-mediated strand displacement logic circuits with toehold-free DNA

2021

DNA nanotechnology, and DNA computing in particular, has grown extensively over the past decade to end with a variety of functional stable structures and dynamic circuits. However, the use as designer elements of regular DNA pieces, perfectly complementary double strands, has remained elusive. Here, we report the exploitation of CRISPR-Cas systems to engineer logic circuits based on isothermal strand displacement that perform with toehold-free double-stranded DNA. We designed and implemented molecular converters for signal detection and amplification, showing good interoperability between enzymatic and nonenzymatic processes. Overall, these results contribute to enlarge the repertoire of su…

Engineering CRISPR guide RNA riboswitches for in vivo applications

2019

CRISPR-based genome editing provides a simple and scalable toolbox for a variety of therapeutic and biotechnology applications. Whilst the fundamental properties of CRISPR proved easily transferable from the native prokaryotic hosts to eukaryotic and multicellular organisms, the tight control of the CRISPR-editing activity remains a major challenge. Here we summarise recent developments of CRISPR and riboswitch technologies and recommend novel functionalised synthetic-gRNA (sgRNA) designs to achieve inducible and spatiotemporal regulation of CRISPR-based genetic editors in response to cellular or extracellular stimuli. We believe that future advances of these tools will have major implicati…

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2.

2018

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP…

A Comparison of Techniques to Evaluate the Effectiveness of Genome Editing

2018

Genome editing using engineered nucleases (meganucleases, zinc finger nucleases, transcription activator-like effector nucleases) has created many recent breakthroughs. Prescreening for efficiency and specificity is a critical step prior to using any newly designed genome editing tool for experimental purposes. The current standard screening methods of evaluation are based on DNA sequencing or use mismatch-sensitive endonucleases. They can be time-consuming and costly or lack reproducibility. Here, we review and critically compare standard techniques with those more recently developed in terms of reliability, time, cost, and ease of use.

Contribution of allelic imbalance to colorectal cancer

2018

Point mutations in cancer have been extensively studied but chromosomal gains and losses have been more challenging to interpret due to their unspecific nature. Here we examine high-resolution allelic imbalance (AI) landscape in 1699 colorectal cancers, 256 of which have been whole-genome sequenced (WGSed). The imbalances pinpoint 38 genes as plausible AI targets based on previous knowledge. Unbiased CRISPR-Cas9 knockout and activation screens identified in total 79 genes within AI peaks regulating cell growth. Genetic and functional data implicate loss of TP53 as a sufficient driver of AI. The WGS highlights an influence of copy number aberrations on the rate of detected somatic point muta…

Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7

2020

This article belongs to the Section Bacterial Viruses.

2017

Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-…

2019

The possibility to modify gut bacterial flora has become an important goal, and various approaches are used to achieve desirable communities. However, the genetic engineering of existing microbes in the gut, which are already compatible with the rest of the community and host immune system, has not received much attention. Here, we discuss and experimentally evaluate the possibility to use modified and mobilizable CRISPR-Cas9-endocing plasmid as a tool to induce changes in bacterial communities. This plasmid system (briefly midbiotic) is delivered from bacterial vector into target bacteria via conjugation. Compared to, for example, bacteriophage-based applications, the benefits of conjugati…

Long-term genomic coevolution of host-parasite interaction in the natural environment

2017

Antagonistic coevolution of parasite infectivity and host resistance may alter the biological functionality of species, yet these dynamics in nature are still poorly understood. Here we show the molecular details of a long-term phage–bacterium arms race in the environment. Bacteria (Flavobacterium columnare) are generally resistant to phages from the past and susceptible to phages isolated in years after bacterial isolation. Bacterial resistance selects for increased phage infectivity and host range, which is also associated with expansion of phage genome size. We identified two CRISPR loci in the bacterial host: a type II-C locus and a type VI-B locus. While maintaining a core set of conse…