TINY TnpB

TAG: GS 3: SCIENCE AND TECHNOLOGY

THE CONTEXT: A breakthrough in genome editing has been achieved with the introduction of TnpB, a smaller yet highly effective tool for plant genome editing. This development promises to overcome the limitations posed by the bulkiness of existing genome editing proteins.

EXPLANATION:

• Genome editing represents a revolutionary scientific breakthrough, enabling precise modifications to the genetic code that dictates an organism’s traits and behaviors.
• This transformative technology has profound applications across biotechnology, human therapeutics, and agriculture, allowing us to develop desired traits and eliminate undesirable ones in various organisms, including microbes, animals, and plants.

Limitations of Current Genome Editing Tools

• Currently, the most widely used proteins for genome editing are Cas9 and Cas12a.
• These proteins function as molecular scissors, cutting and editing DNA.
• However, their significant size, comprising 1,000–1,350 amino acids, poses a challenge for efficient delivery into cells where genetic material is located.
• Advanced editing technologies like base editing and prime editing further increase the bulkiness by requiring the fusion of additional proteins with Cas9 and Cas12a.

TnpB

• A breakthrough in genome editing has been achieved with the introduction of TnpB, a smaller yet highly effective tool for plant genome editing.
• This development promises to overcome the limitations posed by the bulkiness of existing genome editing proteins.
• TnpB proteins are transposon-associated nucleases guided by RNA, considered the evolutionary ancestors of Cas12 nucleases.
• Functionally similar to Cas12a but much more compact, TnpB proteins range from 350 to 500 amino acids, roughly one-third the size of Cas9 and Cas12a.
• For instance, the TnpB nuclease derived from Deinococcus radiodurans, known for its extreme environmental resilience, is only 408 amino acids long.
• Mechanism of Action
  • TnpB uses a short RNA guide to target specific DNA sequences.
  • Upon binding to the target, TnpB cleaves both strands of DNA, leading to insertions or deletions of DNA letters when the cell repairs the broken ends.
  • This modification process alters the genetic sequence.
  • A crucial aspect of TnpB’s specificity is the requirement for a Transposon Associated Motif (TAM) sequence adjacent to the target DNA.
  • For D. radiodurans TnpB, the TAM sequence is TTGAT, allowing access to genomic loci that Cas9 cannot reach.

Repurposing TnpB for Plant Genome Editing

• Optimization for Plant Systems
  • To utilize TnpB for plant genome editing, researchers codon-optimized the TnpB protein sequence and optimized regulatory elements to produce sufficient guide RNA for high-efficiency editing.
  • Through testing in rice protoplasts, four different genome editing vector systems were evaluated, identifying the most effective version for monocot plants like rice.
• Adaptation for Dicots
  • Given that genome editing systems effective in monocots may not perform well in dicots, researchers also developed dicot-specific TnpB vectors.
  • Successful editing was demonstrated in Arabidopsis, where deletions predominantly occurred at the target loci in both rice and Arabidopsis.
  • This capability makes TnpB suitable for disrupting gene functions, which is essential for applications such as removing antinutrient factors, enhancing nutrient content, and improving stress resistance.

Advanced Applications of TnpB

• Researchers have developed a deactivated form of TnpB (dTnpB), which, while unable to cut DNA, can still bind to target sequences specified by guide RNA.
• Fusing dTnpB with additional proteins can recruit factors that activate genes, thereby enhancing gene function.
• This activation tool has the potential to boost gene activity, paving the way for developing superior crops.
• Another advanced application involves fusing dTnpB with proteins capable of altering single DNA letters.
• This precise editing tool allows for the correction of specific genetic sequences, enabling crop innovation at the genetic code level with single-letter accuracy.

Future Prospects

• Enhancing Crop Traits
  • Researchers are leveraging TnpB to create rice plants with improved yields and enhanced climate resilience.
  • The versatility and effectiveness of TnpB position it as a promising tool for plant genome engineering.
  • The technology is expected to be adopted by plant biologists, biotechnologists, and breeders for various crops, driving agricultural advancements.

SOURCE: https://phys.org/news/2024-07-tiny-tnpb-generation-genome-tool.html