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"A new engineered gene editing system, proven in bacteria and with promise for use in plants, animals and humans, addresses some of the limitations of current gene editing tools with the hope of improving their applications for research, for better crops or to treat diseases. ...
A new study describes how scientists fused together components in bacteria that do not naturally occur together, thereby creating the potential for important design opportunities that the group is currently exploring. These components include RNA-guided enzymes believed to represent the evolutionary origins of CRISPR-Cas systems. ...
Previous gene editing methods enabled researchers to change a single base pair of DNA (called base editing) or even a few of them (called prime editing), while this new technique joins a group of new technologies that can introduce whole sections of DNA to correct a disorder without complications. That’s important, because many diseases involve stretches of DNA, making it necessary to change large sections of DNA at once. ..."
From the highlights and abstract:
"Highlights
• TnpB-family proteins can be engineered for RNA-guided transposition
• TniQ-TldR fusion systems achieve efficient, unidirectional DNA insertion
• TniQ-TldR fusions allow a minimal, three-component, programmable integration system
• Changes at the TniQ-TnsC interface in wild-type V-K CAST attenuate off-site insertion
Summary
Programmable DNA integration using CRISPR-associated transposase elements (CASTs) offers powerful capabilities for genome engineering. The large single effector Cas12k CAST examples evolved from a minimal TnpB nuclease protein. Here, we engineer a de novo RNA-guided transposition systems in bacteria, where the single guide RNA effector components are repurposed nuclease-dead TnpB-family proteins.
These compact systems mediate high-efficiency guide-RNA-directed DNA insertion with preserved orientation control, target immunity, and release of a host factor requirement and can be paired with an exonuclease domain to mediate cut-and-paste transposition.
In this engineered context, the TnpB derivatives show features not predicted from the original enzymes, suggesting untapped avenues for improvement.
In parallel, we show that mutations at the TniQ-TnsC interface in the Cas12k CAST system selectively attenuate off-site insertions while enhancing on-site activity.
These results establish how Cas12 proteins and antecedent TnpB proteins can be engineered for high performance and specificity with guide-RNA-directed systems."
Graphical abstract
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