Transposons Help Expand the CRISPR Toolbox: Another Highly Efficient Orthogonal CAST System CRISPR RNA-guided insertion of large 10 kb DNA fragments

The CRISPR-Cas system in prokaryotes can guide the degradation of exogenous genetic elements such as bacteriophages and plasmids through RNA, enabling DNA-encoding, RNA-mediated, nucleic acid-targeted adaptive immune systems
.
In addition, the CRISPR-Cas system is also involved in a DNA transposon-like (guided by CRISPR RNA).

Inspired by it, discerning researchers rushed to try to develop it into a new genome engineering tool, the CRISPR-associated transposon (CAST), a system that combines CRISPR-Cas and transposon proteins that can be used for programmable site-specific integration of fairly large "cargo DNA," avoiding the need
for DNA cleavage and homologous-directed repair involving endogenous repair mechanisms.

Several different types of CRISPR-associated transposons (casts) have been identified, including: I-F3, V-K, and I-B1 and I-B2
。 The I-F3 and I-B systems utilize the Cascade (CRISPR-associated complex for antiviral defense) effector complex, and are naturally deficient Cas3 helicase-nuclease typically responsible for DNA cleavage in type I systems, while V-K systems utilize Cas12k
, which naturally lacks nuclease function 。 All three CAST systems use a combination of the CRISPR-Cas mechanism and transposon proteins for RNA-guided DNA transposon, similar to Tn7
.
I-F3 CAST Tn6677 has been simplified to a single plasmid form and used in multiplex genome engineering, as well as species- and site-specific genome editing in the context of mixed communities, a genome engineering tool that offers great promise
for site-specific integration with large DNA payloads.

Although there are already some simplified systems in place to achieve complex and multiplex genome engineering while avoiding problems such as target site immunity and transposon recombination, researchers at North Carolina State University in this study attempt to develop an efficient and orthogonal I-F3 CAST extended genome editing tool
。 They selected the I-F3 CRISPR-related transposon system derived from γ Proteobacteria, analyzed all components critical for transposable activity, tried to analyze their RNA-guided DNA integration activity at different temperatures, different transposon sizes, and different target PAM sequences, and performed functional tests in E.
coli, enabling large fragments of DNA with up to 10,000 extra bases to the E.
coli genome, rewriting rather than just editing large fragments of their genome
。 The results show that the new CAST system has significant integration efficiency
in terms of temperature, transposon size, and flexible PAM requirements.
The findings support the classification of these CAST systems into functional compatibility groups for efficient and orthogonal RNA-guided DNA integration
.
Using a new, CRISPR RNA-guided DNA integration system, this work expands the CRISPR-based toolbox that can be applied to complex and wide-ranging genome engineering efforts that could have a significant impact
on the manipulation of bacteria and other organisms when flexible genome editing is required in therapeutics, biotechnology, and more sustainable and efficient agriculture.

Bacteria use CRISPR-Cas as an adaptive immune system to fend off attacks from enemies such as viruses
.
These systems have been applied by scientists to remove, cut, and replace specific genetic code sequences
in various organisms.
The new findings suggest an exponential increase in the amount of genetic code that can be moved or added, which could increase the functionality of
CRISPR.

"In nature, transposons chose the CRISPR system to selfishly move themselves through the genome of an organism to help themselves survive
.
In turn, by integrating a programmable CRISPR-Cas system with the transposon, we can "move" the "genetic cargo" that we design to perform certain functions, "similar to bringing specific luggage or cargo into a car and delivering the loaded cargo when the car arrives at its destination," said Rodolphe Barrangou, a Todd R.
Klaenhammer Visiting Professor of Food, Bioprocessing and Nutritional Sciences at North Carolina State University.
Also the corresponding author
of this paper.
Barrangou said: "Using this method, we demonstrated that we can design genomes
by moving large segments of DNA up to 10,000 bp.
" "Nature has done this – bioinformatics data shows that transposon-based CRISPR systems can move up to 100,000 genetic letters, and now we can control and engineer it
by using this system.
"

The researchers demonstrated the effectiveness of
this approach both in vitro and in E.
coli.
The researchers selected 10 different CRISPR-related transposons to test the effectiveness of
the method.
This method is suitable for all 10 transposons, although their effectiveness varies
depending on factors such as temperature and transposon load size.

Avery Roberts, a graduate student at North Carolina State University and first author of the study, said: "It's exciting that all the systems we tested worked after
reconstructing them from protist morphology into genome editing tools.
" "We are discovering new features in these systems, but as the field rapidly evolves, more relevant discoveries and applications may emerge.
"

Studies have also shown that this method can be used for different transposons
at the same time.

Barrangou said: "Unlike other CRISPR systems, such as the familiar type II Cas-9 system, which can only carry one gene, we can introduce the entire metabolic pathway, integrating a whole new set of functions into the organism
.
" "In the future, this could mean providing plants with more flexible disease resistance or drought resistance
.
"

"We are excited about these findings and see the potential to apply these newly discovered systems to crop crops to accelerate the development of more resilient, higher-yielding varieties
," said Gusui Wu, global head of seed research at Syngenta Seeds.

The paper was published in Nucleic Acids Research
.
The funding was provided by Syngenta Seeds, which was acquired
by ChemChina in 2017.
Co-authors of the paper include Avery Roberts, a graduate student at North Carolina State University, and Matthew Netseny
, a former doctoral student at North Carolina State University.