Genetic “hitchhikers” can be targeted with CRISPR

Nucleic acid research (2022). DOI: 10.1093/nar/gkac985″ width=”800″ height=”398″/>

Form and function of CRISPR-associated type I-F3 transposons. (A) Tn7479 is shown as a representative type I-F3 CAST. Genes essential for transposition, cargo genes not essential for transposition, the att site (rsmJ), and the ends of the transposon (R and L) are indicated. The atypical CRISPR array repeat is colored light gray. (B) Detailed view of the transposon ends and the Tn7479 CRISPR array in (A). Eight putative TnsB binding site sequences were used to generate a consensus WebLogo (top). Duplication of target sites with a length of 5 bp. (black), indicative of Tn7-like transposition events, and the terminal inverted repeats (green) that define the ends of the transposon are shown next to TnsB binding sites (purple arrows) at the ends of the transposon. The CRISPR array contains a self-targeting spacer that is complementary to the att (rsmJ) site region and is flanked by an atypical repeat (light gray diamond). The rsmJ target pad and homing pad have inconsistencies colored red. (C) Mechanistic overview of type I-F3 CRISPR RNA-directed DNA transposition. The TniQ-Cascade complex is directed to a target site complementary to the spacer sequence of the bound cRNA. 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 transposon integration of ∼50 bp. downstream from the end of the target site. (D) A phylogenetic tree of representative TnsB clades is shown with Tn7 as an outgroup and eleven diverse CAST type I-F3 selected for characterization. (E) The cargo genes of the eleven I-F3 CAST types selected for characterization were used to generate a heat map depicting the number of genes for each category of clusters of orthologous genes (COGs). credit: Nucleic acid research (2022). DOI: 10.1093/nar/gkac985

In a new study, North Carolina State University researchers characterize a set of molecular tools for rewriting, not just editing, large stretches of an organism’s DNA based on CRISPR-Cas systems linked to selfish genetic “autopilots” called transposons.

Researchers are exploring different IF CRISPR-Cas systems and engineering them to add genetic cargo—up to 10,000 extra letters of genetic code—to the transposon cargo to make the desired changes in a bacterium—in this case, E. coli. The paper appears in Nucleic acid research.

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

Bacteria use CRISPR-Cas as an adaptive immune system to resist attacks from enemies such as viruses. These systems have been adapted by scientists to delete or cut out and replace certain sequences of the genetic code in different organisms. The new finding shows that exponentially more 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 themselves survive. We in turn co-opt what happens in nature by integrating with transposons a programmable CRISPR-Cas system that can move genetic cargo that we engineer to perform a specific function,” said Rodolphe Barango, emeritus professor of food, bioprocessing and of Nutrition at North Carolina State and corresponding author of the paper describing the study.

“Using this method, we have shown that we can create genomes by moving pieces of DNA up to 10,000 letters long,” Barangu 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 with this system.

“To complete the hitchhiking analogy, we design the hitchhiker to deliver a particular piece of luggage or cargo to the car to deliver some type of payload when the car arrives at its destination.”

The study shows that the researchers are 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. This approach worked with all 10 transposons, although their effectiveness varied depending on factors such as temperature and size transposon‘s cargo load.

“It was interesting to find that all the systems we tested were functional after being reconstructed into genome-editing tools from their native biological forms,” ​​said Avery Roberts, a graduate student at NC State and first author of the study. “We have discovered new possibilities for these systems, but there are likely to be many more related discoveries and applications as the field moves at a rapid pace.”

The study also showed that the method can be used with different transposons simultaneously.

“Instead of a single gene—as is the case with other CRISPR systems, such as the more common type II Cas-9 system—we can introduce an entire metabolic pathway to enable a whole new set of functions in the body,” Barangu said. “In the future, this could mean, for example, giving plants more flexible resistance to disease or drought.”

“We are excited by these findings and see the potential to apply these newly discovered systems in agricultural plants to accelerate the development of more resistant and high-yielding varieties,” said Gusui Wu, global head of seed research at Syngenta Seeds.

Barangou and Wu add that the work in this research provides an excellent example of a public-private partnership that drives scientific discovery and trains tomorrow’s workforce.

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

Additional information:
Avery Roberts et al., Functional Characterization of Different Types of IF CRISPR Transposons, Nucleic acid research (2022). DOI: 10.1093/nar/gkac985

Citation: Genetic hitchhikers can be controlled with CRISPR (21 November 2022), Retrieved 21 November 2022 from

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