Interesting question. We have debated this issue earlier on this platform...

This question is under active research

Hi Ivan Tjahja Pranata,

1. The nif genes are found in both free-living nitrogen-fixing bacteria and in symbiotic bacteria associated with various plants. The nif genes are 17 genes (Nif regulon) encoding enzymes involved in the fixation of atmospheric nitrogen into a form of nitrogen available to living organisms (ex. plants).

2. So, if you want the rice plants can fix N2 on their own and provide fertilizer for themselves, you should transfer those bacterial genes (17) or Nif regulon into the rice genome.

3. Transferring 17 genes is not easy, but it has been tested in plants and reported. And researchers have suggested that the organelles (ex. chloroplasts and mitochondria) are the right places to host those genes. See attached two papers, one is for chloroplasts and the other is for mitochondria

4. CRISPR/Cas9 is more efficient in using for knocking out genes, instead of knocking in genes.

(edited on 2/28/2018)

Hi Ivan Tjahja Pranata,

The applications of using CRISPR-Cas9 is used to generate a DBS at a specific locus for an allele is: (1) just to knock out the allele, or (2) to knock-in a gene cassette at the DSB site through homologus recombination (HR), with a donor plasmid (see attached figure). In the figure, on the left-handed side, it indicates a locus is disrupt, and on the right-handed side, a gene cassette is integrated at the DSB site. However, it is with much higher efficiency to just knock out the allele. Using homologus recombination to integrate a gene cassette is not as efficient (see attached paper).

So, it is possible to integrate the Nif genes into rice genome (at a specific genomic locus) through using CRISPR/Cas9 technology. However, how to increase the HR rate in the cells and make integration more efficiently done is an important taskin the future. Scientists are working hard to find methods to increase the HR rate. I will post some references here later.

Here, it should be a good paper to discuss how to deliver genes into plants cells (knock in) in plants. I cannot access to this paper. If you can, please send me a copy. Apprciated!!

Title:

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens.

Methods. 2017 May 15;121-122:103-117. doi: 10.1016/j.ymeth.2017.04.024. Epub 2017 May 4.

Abstract Beyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.

Many researchers proposed similar theories for manipulating C3 genomes to be C4, and confer nitrogen fixation property to Poaceae members. As Yuan-Yeu said, its not easy to confer more than one gene!

The integration of C4 photosynthesis system into C3 plant is much more difficult than just transfer 16 genes into a plant genome. The conversion of C3 crops, such as rice, towards C4 photosynthesis is extremely difficult is partially because the C4 carbon-concentrating biochemical cycle spans two cell types (Kranz anatomy) and thus requires specialized anatomy (see attached figure). Transfer of more than 10 genes into plants is not as sophisticated and have been done and reported, although the work can be tedious.