ICAR, Penn State team creates device small enough to edit plant genomes

Flour, chocolate, cocoa powder, eggs and butter are all ingredients that are useful for making your desired dessert.

All you need at this point is a step-by-step recipe that will help you make delicious brownies.

much bigger than its limit

Nature also has the ingredients needed to ‘make’ living organisms, using a genetic instruction book called the genome. A small change in the structure of the genome can determine whether the organism being created is a flower that displays two petals, a cat with large or small ears, or whether coriander leaves will taste like soap to some people.

with the help of The gene-editing tool CRISPRToday scientists can precisely edit genomes to incorporate desired genetic traits or remove undesirable traits.

CRISPR has the potential to revolutionise agriculture, particularly by allowing agricultural scientists to increase crop yields and improve resistance to disease and abnormal weather through gene-editing. However, there has been a serious obstacle: a commonly used form of the CRISPR system is too large for plant genomes.

This system uses one of two proteins, Cas9 or Cas12, to target specific parts of DNA. But they are too bulky for plant cells.

Smaller is better

A team of researchers led by Qutubuddin Molla of the ICAR-National Rice Research Institute, Cuttack, and Mirza Beg of the Pennsylvania State University, US, has presented an alternative that could solve this major problem in plant genome editing. Journal of Plant Biotechnology,

They reported developing a plant genome editor that incorporates a protein called ISDra2TnpB, derived from bacteria called Bacteroidetes. deinococcus radiodurans (Famous for being able to survive extreme environmental conditions.) ISDra2TnpB is less than half the size of Cas9 and Cas12.

V.S. Shresthi Tavva, principal scientist at the crop improvement programme at the Tata Institute for Genetics and Society (TIGS), Bengaluru, who was not involved in the study, expressed excitement at the findings.

“Right now, (since) there are not many options available for plant genome editors, the improved TnpB definitely adds value. We should take advantage of the size of TnpB to create edited plants for various traits of interest,” he said.

Editing skills of TNPB

TnpB is a protein made up of about 400 amino acid units (different combinations of 20 amino acids make up all proteins). It belongs to the family of transposable elements, or transposons. Sometimes called “jumping genes,” transposons are parts of the genome that can move from one location to another.

The genome consists of two strands of DNA that are connected to each other. Each strand is made up of building blocks called nucleotides. In turn, each nucleotide consists of three fragments; two of them are the same in all while the identity of the third can be one of four options: adenine (A), thymine (T), cytosine (C) or guanine (G). The ‘sequence’ of DNA refers to the order in which the nucleotides containing these four compounds are arranged.

In the new system, TnpB rides on a piece of RNA that carries it to the target DNA sequence. Once there, TnpB binds to the sequence and cuts it off. The cell that contains this DNA repairs the cut by restoring the ‘correct’ sequence. Thus, the genome is modified to replace an undesirable sequence with a desirable one.

The researchers behind the new study achieved 33.58% editing efficiency in the genome of an average plant using the genome editing capabilities of the TnpB-based system, on targets that Cas9 or Cas12 could not reach. They demonstrated that the genome editor was effective on both types of flowering plants – monocots (such as rice, which has a single seed leaf) and dicots (such as ArabidopsisA plant related to cabbage and mustard having two seed leaves).

Codons and regulators

The team also created four versions of the TnpB-based editing tool and tested them on rice protoplasts – plant cells without cell walls – to identify the best among them. In their initial experiments, the versions had low editing efficiency.

To improve this, Dr. Molla and his colleagues did two things. First, they used a process called codon optimization. For example, cells in the body make the amino acid lysine by following an instruction in the genome represented by a sequence of three nucleotides. Such sequences of three are called codons.

The codon sequence containing the recipe for lysine varies in different organisms. TnpB is a protein extracted from D. radioduransA prokaryotic bacteria, which has a different codon for lysine than eukaryotes such as plants. So the researchers edited the codon bias of TnpB to match that of rice protoplasts to improve editing efficiency, Dr. Molla explained.

The second thing the researchers changed was the regulatory elements. When TnpB and the specific RNA that carries it to the target DNA are transferred from a prokaryote to a eukaryote, the researchers also need to include sequences called promoters and terminators that control and regulate the expression of TnpB.

“We have added promoters that can increase the expression of TnpB and provide better editing,” Dr. Molla said.

A high-resolution upgrade

The researchers have completed finalizing the TnpB-based gene-editing system. They inactivated TnpB and combined it with another protein to create a ‘hybrid’ base editor.

With the guide RNA, this editor can change a single nucleotide in the DNA sequence.

This was not possible in the previous version with active TnpB, as it only deleted DNA sequences and could not replace one sequence with another.

Thus, the new base editor opens up exciting possibilities for crop innovation by facilitating changes in genes at the level of individual nucleotides.

The future of edited plants

The TnpB-based editors built by the researchers can edit plant genomes using both base editing and transcription activation, two techniques widely used in plant synthetic biology.

Dr. TavvaHowever, he said most of the claims were based on data obtained from protoplasts and the scenario may change when it comes to the process of an organism absorbing foreign DNA and integrating it into its genome.