Genetic 'hitchhikers' can be navigated using CRISPR

Genetic ‘hitchhikers’ can be navigated using CRISPR

Genetic 'hitchhikers' can be directed using CRISPR Form and function of CRISPR type I-F3 associated transposons. (A) Tn7479 is shown as a representative type I-F3 CAST. The genes essential for transposition, the cargo genes not essential for transposition, the att site (rsmJ) and the ends of the transposon (R and L) are marked. The atypical repeat of the CRISPR network is colored in light gray. (B) A detailed view of the transposon ends and the CRISPR array of Tn7479 in (A). The eight putative TnsB binding site sequences were used to generate a consensus WebLogo (top). 5 bp target site duplication events (black) indicative of Tn7-like transposition events and terminal inverted repeats (green) that define transposon ends are shown next to TnsB binding sites (purple arrows) in the ends of the transposon. The CRISPR network contains a self-targeting spacer that is complementary to a region of the att site (rsmJ) and flanked by an atypical repeat (light gray diamond). The rsmJ target site and the self-targeting spacer possess offsets colored in red. (C) A mechanistic overview of type I-F3 CRISPR RNA-guided DNA transposition. The TniQ-Cascade complex is guided to the target site complementary to the bound cRNA spacer sequence. TnsB proteins bind to sites present at the ends of the transposon and transposition, regulated by TnsC activity between the TniQ-Cascade complex and the heteromeric transposase TnsAB, results in the integration of the transposon approximately 50 bp downstream of the end. of the target site. (D) A phylogenetic tree of representative TnsB clades is shown with Tn7 as an outgroup and eleven diverse type I-F3 CASTs selected for characterization. (E) The cargo genes of the eleven type I-F3 CASTs selected for characterization were used to generate a heat map displaying the number of genes for each category of orthologous gene groups (COGs). Credit: Nucleic acid research (2022). DOI: 10.1093/nar/gkac985

In a new study, researchers at North Carolina State University characterize a range of molecular tools for rewriting – not just editing – large chunks of an organism’s DNA, based on CRISPR-Cas systems paired with “auto Selfish genetic “stoppers” called transposons.

Researchers are studying various type IF CRISPR-Cas systems and designing them to add genetic cargo – up to 10,000 additional letters of genetic code – to the transposon cargo to make desired changes to a bacterium – in this case, E .coli. The paper appears in Nucleic acid research.

The results expand the CRISPR toolbox and could have significant implications in manipulating bacteria and other organisms at a time when the need for flexible genome editing in therapeutics, biotechnology and more sustainable and efficient agriculture is necessary.

Bacteria use CRISPR-Cas as an adaptive immune system to resist attacks from enemies like viruses. These systems have been adapted by scientists to delete or cut and replace specific genetic code sequences in a variety of organisms. The new discovery shows that exponentially greater amounts of genetic code can be moved or added, potentially increasing the functionality of CRISPR.

“In nature, transposons have co-opted CRISPR systems to selfishly move around an organism’s genome to help itself survive. We in turn co-opt what happens in nature by integrating into transposons a CRISPR- Programmable case that can move genetic cargo that we engineer to perform certain functions,” said Rodolphe Barrangou, Todd R. Klaenhammer Professor Emeritus of Food, Bioprocessing, and Nutrition Sciences at NC State and corresponding author of an article describing the research.

“Using this method, we have shown that we can design genomes by moving pieces of DNA up to 10,000 letters long,” Barrangou said. “Nature already does this – bioinformatics data shows examples of up to 100,000 genetic letters being moved by transposon-based CRISPR systems – but now we can control and engineer it using this system.

“To complete the hitchhiking analogy, we design the hitchhiker to bring certain luggage or goods into the car to deliver some type of payload when the car arrives at its destination.”

The study shows researchers proving the effectiveness of the method both in vitro on the lab bench and in vivo on E. coli. The researchers selected 10 different transposons associated with CRISPR to test the effectiveness of the method. The approach worked with all 10 transposons, although their efficiency varied depending on factors such as temperature and the size of the transposon’s loading charge.

“It was exciting to see that all of the systems we tested were functional after reconstructing them into genome editing tools from their native biological forms,” ​​said Avery Roberts, NC State graduate student and first author. of the study. “We have discovered new features of these systems, but there are likely many more relevant discoveries and applications to come as the field evolves at a rapid pace.”

The research also showed that the method could be used with different transposons at the same time.

“Instead of a single gene, as is the case with other CRISPR systems like the more familiar Cas-9 type II system, we can integrate an entire metabolic pathway to incorporate a whole new set of functions into a organism,” Barrangou said. “In the future, this could mean providing plants with more flexible resistance to disease or drought, for example.”

“We are excited about these findings and see the potential for applying these newly discovered systems in crop plants to accelerate the development of more resistant and higher yielding varieties,” said Gusui Wu, Global Head of Plants Research. seeds at Syngenta Seeds.

Barrangou and Wu add that the work in this study provides an excellent example of public-private partnerships that are driving scientific discovery and training the workforce of tomorrow.

Co-authors of the paper include NC State graduate student Avery Roberts and former NC State Ph.D. student Matthew Nethery.

More information:
Avery Roberts et al, Functional characterization of various transposons associated with IF CRISPR, Nucleic acid research (2022). DOI: 10.1093/nar/gkac985

Provided by North Carolina State University

Quote: Genetic Hitchhikers Can Be Navigated Using CRISPR (2022, Nov 21) Retrieved Nov 21, 2022 from

This document is subject to copyright. Except for fair use for purposes of private study or research, no part may be reproduced without written permission. The content is provided for information only.

#Genetic #hitchhikers #navigated #CRISPR

Leave a Comment

Your email address will not be published. Required fields are marked *