Can indigenous efforts at genome-edited crop breeding offer food security?

Against this backdrop, Kutubuddin Ali Molla and his team at the Indian Council of Agricultural Research’s Central Rice Research Institute, Cuttack, offer renewed hope. They have successfully demonstrated that RNA-guided transposon associated protein (TpnB) – which is much more compact than Cas-proteins and hence easier to deliver – can perform genome editing in rice plants by introducing mutations that interfere with gene expression levels. Let’s understand the basics of this technology.

What are transposons, the jumping genes

Chromosome number and genome size vary dramatically across species. For example, a deer species has only six chromosomes, humans have 46, chimpanzees 48, and the fern Ophioglossum reticulatum has an extraordinary 1440. Genome size also varies widely and does not directly reflect organismal complexity. For example, the human genome is about 200 times larger than that of unicellular yeast, yet 30 times smaller than that of some amphibians and plants; some single-celled amoebae even possess far more DNA than humans. 

Despite these differences, humans and chimpanzees share nearly 99 per cent DNA sequence identity and about 80 per cent similarity in functional proteins. This highlights a key principle of biology: complexity depends more on how genetic material is organised and regulated than on how much DNA is present. This is exactly where transposons come into the picture. 

The genomes of organisms are highly disordered, exceptionally long sequences. Over billions of years of evolution, almost none of the genetic material has ever been discarded. Relics of troublesome pasts are present in our genomes. The pathogenic viral DNAs has been tamed and pieces of foreign DNA have been incorporated. Some sequences are very much alive and are still moving or jumping as their inventor, Nobel Laureate Barbara McClintock, called them “jumping genes”, now known as transposons. 

Far from being rare, transposons make up over 50 per cent of the human and maize genomes, while only about 2 per cent of the human genome directly encodes proteins. There are three types of transposons: DNA type, retroviral or LTR and Poly A. By inserting themselves into new genomic locations, transposons continuously reshape the genome. 

Their movement can cause harmful mutations. For example, insertions of L1 retrotransposon into the factor VIII or APC genes can lead to haemophilia and colon cancer. Transposon activity can be triggered by cellular stress, radiation, and toxic chemicals. However, transposons are not purely destructive. They also contribute to genome evolution by enabling exon shuffling, gene innovation, and even the repair of DNA double-strand breaks. Thus, transposons represent both a source of genetic instability and a powerful engine of evolutionary change.

Cas9 proteins, descendants of transposon-encoded nucleases

Recent work tracing the origin of CRISPR systems shows that Cas9 most likely evolved from a transposon-encoded nuclease called IscB, which belongs to the IS200/IS605 family of transposons and is widely distributed in prokaryotes. IscB and Cas9 share a similar overall domain architecture, including a split RuvC nuclease domain and an HNH endonuclease domain. Evolutionary insights through cluster and phylogenetic analysis showed Cas9s descended from a single ancestral IscB. 

Like Cas9, IscBs are also associated with CRISPR-like arrays that generate non-coding RNAs that bind to the IscB proteins. They contain a single variable terminal domain akin to CRISPR-guide RNA. Thus, IscB has many hallmarks of an RNA-mediated genome editor like the CRISPR-Cas system. 

These complexes can expand their target through RNA switching, insertion of multiple copies of transposon, and complexing with RNAs generated from other distant arrays. Target analysis showed that unlike CRISPR, which primarily defends against the foreign DNAs, RNA guided IscB arrays are primarily for non-defence purposes (or transposon-centric role) and predominantly target their own genomic loci for cleavage.   

Thus, one arm of a general-purpose transposon encoded nuclease evolved into a cellular defence system. IS200/IS605 family transposons commonly carry such RNA-guided nucleases. IscB homologues have been identified in eukaryotes also.For example, multiple iscB loci occur in the chloroplast genome of the terrestrial green alga Ignatius tetrasporus. Functionally, one IscB ortholog, OgeuIscB, has been shown to introduce insertions and deletions (INDELS) at 28 of 46 tested target sites in human HEK293FT cells. This demonstrates that these compact transposon-derived editors can be harnessed for programmable genome editing, yet their natural function remains unknown.

Regulation of jumping genes 

Members of the IS200/IS605 family of transposons carry the TpnA gene, which is essential for transposition. Sometimes these elements can carry the TpnB gene, which putatively regulates the condition and frequency of transposition. Yet, recent studies have shown that compact TpnB encoded by transposon ISDra2 in bacteria can be reprogrammed to cleave target sites in mammalian cells. 

Essential evidence came from biochemical studies where it was established that expression of the entire transposon was required for expression of a complete TpnB protein. Purified TpnB protein is always associated with 150 nucleotide non-coding RNA, suggesting its obligatory association. Further, like Cas9, which requires a (NGG) nucleotide sequence as PAM, TpnB requires TTGAT sequence, also called TAM. 

In vivo experiments also show TpnB can cleave target DNA sequence in bacteria E. coli and human cells like HEK293T (kidney cells). The frequency of INDELS due to DNA cleavage by TpnB and repair by cellular machinery is around 10-20 per cent like CRISPR Cas9 and Cas12 based editing. Deeper analysis revealed dominations of deletions over insertions at the cleavage site- like Cas12 activity. Sequence comparison shows similar domain organisation and split RuvC motif, suggesting links between TpnB and CRISPR-Cas systems. 

Mechanistically, TpnB provides a backup plan for transposons to reinstate them at the original site by making a DNA break that triggers recombination to copy the transposon back to the original site in case transposition at different sites fails. Thus, Cas9 represents a domesticated transposon nuclease adapted for immune defence, and TnpB reveals how mobile elements themselves originally used RNA-guided nucleases for their own genomic survival.

How transposon-based genome editing works in rice plants

The ICAR CRRI team demonstrated a broad spectrum of applications of the TpnB-based genome editing. Due to the compact size of TpnB, a protoplast-based simple delivery workflow achieved 34  per cent editing efficiency. However, the criterion of TAM (target-adjacent motif) requirement rendered only 0.35 per cent of genomic locus feasible for targeting. 

Six locations were targeted after designing specific guide RNAs, and 7 to 53 base pair deletions were observed, which indicate Cas12-like functioning of TpnB across all walks of eukaryotic cells, including plants. A multiplexed editing experiment was also performed to target two genes at different locus simultaneously, and on average, 5 per cent INDELS were observed. 

The team also checked the system on the model dicot plant Arabidopsis and found 0.2-0.4 per cent editing efficiency. However, strong promoters driving high RNA synthesis gave better results. A catalytically dead TpnB mutant D191A was used to achieve base editing rather than DNA cleavage, resulting in very low-level base editing. 

Among other experiments, they also used the system for gene activation and performed expression optimisation. Finally, they targeted genes responsible for chloroplast development in regenerated rice plants to generate albino rice plants through monoallelic and bialleilic editing. The monoallelic editing efficiency was observed to be 38 per cent (20 plants out of 53). These experiments thereby establish the feasibility of transposon-mediated genome editing as an alternative to the CRISPR-Cas system.

Post read questions

What is the biological origin of the CRISPR-Cas9 system, and how does it function as an adaptive immune mechanism in bacteria?

What are transposons, and why did Barbara McClintock refer to them as “jumping genes”? Classify the major types of transposons found in eukaryotic genomes.

What is the difference between genetically modified (GM) crops and genome-edited (GE) crops? In what ways does new gene-editing technology seek to enable cheaper, commercial GE crop breeding and address food security and agricultural needs?”

How does the TnpB-based genome-editing technology developed by the Indian Council of Agricultural Research work, and how is it similar to CRISPR-Cas systems?

(Dr. Arunangshu Das is the Principal Project Scientist at the Centre for Atmospheric Sciences, Indian Institute of Technology, Delhi.)

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