CRISPRSYSTEM

Identification and Characterization of Host and Phage Proteins Interacting with the CRISPR System

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 Obiettivo del progetto (Objective)

'The CRISPR system was recently identified as a bacterial defense mechanism against phages and plasmids. The CRISPR system is composed of DNA arrays containing short sequences identical to those present in phages and plasmids. These short DNAs are transcribed and processed by CRISPR associated proteins that also guide other CRISPR proteins to target the invading DNA. Only a few of the CRISPR components have been characterized to date and their mechanism of action is still largely unknown. Phage defense mechanisms probably have co-evolved against the CRISPR system, but none have yet been found. We propose to identify phage genes that counteract the CRISPR system. We will utilize screens that make use of the bacterial version of the yeast two-hybrid genetic system, phage genomic libraries, and biochemical assays to identify phage genes that help phages evade the CRISPR system. The candidate genes will be characterized both genetically and biochemically to allow structural studies of their interactions with the CRISPR system. These genes will then be cloned into commercial phages that are used to kill bacterial pathogens so that the phages acquire resistance to the CRISPR defense mechanism. We also propose to identify the yet unknown E. coli proteins that participate in the activity of the CRISPR system by using genetic screens of transposon insertion mutant libraries. The identification of novel proteins that participate in the CRISPR system will constitute a significant step toward the ultimate goal of reconstituting the complete system from purified proteins. Finally, we propose to clone all the components of the E. coli CRISPR system and render them functional in heterologous prokaryotic organisms such as attenuated strains of Salmonella, Shigella and more distantly related species such as Streptococci. Cloning the genes of the E. coli CRISPR system into heterologous bacteria will allow us to genetically address the role of each gene.'

Introduzione (Teaser)

The recent breakthrough applications of the CRISPR/Cas bacterial immune system urged European scientists to study its features in-depth.

Descrizione progetto (Article)

A recent discovery unveiled the existence of a prokaryotic immune defence mechanism that confers resistance to foreign genetic elements such as phages. The so-called CRISPR/Cas system is based on the recognition of specific DNA sequences known as CRISPRs (clustered regularly interspaced short palindromic repeats) by the CAS nucleases.

Researchers realised the power of the system at gene editing and have successfully utilised it to genetically manipulate human DNA. As a result, the CRISPR/Cas system holds great prospects for human health, as well as in numerous biotechnological applications.

The EU-funded 'Identification and characterization of host and phage proteins interacting with the CRISPR system' (CRISPRSYSTEM) project focused on delineating basic aspects of the system. Using an experimental platform established in the host lab, project scientists studied the CRISPR adaptation process. Their work aimed to identify which proteins, DNA elements and steps are required for the process to work.

They discovered a novel element in sequences acquired to confer immunity against bacteriophages. This element enhanced the acquisition of these sequences into the CRISPR array. In addition, a new protein for the system's immune function was found.

Furthermore, the project showed that the activity of the CRISPR/Cas system extends to confer protection against lysogens and prophages. This led the research team to develop a new platform for modifying bacteriophage genomes using the CRISPR/Cas system.

Taken together, the activities of CRISPRSYSTEM significantly advanced knowledge on the recently described bacterial CRISPR/Cas system. Given the system's promising applications, the new information opens up more avenues of exploitation of this revolutionary approach.