Dr. Prabhjit Chadha-Mohanty
Dr. Inez H. Slamet-Loedin

To increase frequency of targeted modification in rice using Geminiviral vectors for delivery of the nucleases (ZFN, TALENs and CRISPR/CAS) and the donor to enhance homologous recombination-mediated gene targeting at a reporter locus in rice. To achieve this main objective we will:
(i) transform site-specific integration donor plasmids constructs, containing the GFP marker gene, for the purpose of gene integration and mutation induction;
(ii) develop the Nipponbare and/or lndica lines containing the TALENs and/or CRISPR/Cas platform at lRRl;
(iii) test the vectors containing elements from Bean yellow dwarf virus, Tomato leaf curl and Wheat dwarf virus for effective delivery of qenes in rice.

Appropriate biosafety and phytosanitary import regulations will be followed. Transgenic and untransformed control plants will be grown in CL4 greenhouses and biosafety screenhouse. Measures implemented were also previously approved in DOST-BC2011-0258. Biosafety measures that will be taken: · All vectors will be kept in the plasmid collection of Genetic transformation lab, PBGB, lRRl Los Banos after receiving · The tested plants will be kept in isolation in the CL4 transgenic greenhouse to prevent access of unauthorized personnel. · Plant materials for disposal will be placed in sealed plastic bags. · Plants grown in CL 4 will be destroyed and the pots and soil will be autoclaved at high temperature and high pressure. · Plant materials that will be grown and evaluated in biosafety screenhouse will be disposed through appropriate disposal system in compliance with biosafety and related regulations in the Philippines. · Access to screenhouse is for authorized staff only. · A barcode entry system is applied in the screenhouse to record the entry of personnel; a log book is available for visitors not having lRRl access card system. · Screenhouse facilities are adequate to prevent entry of rats and birds. · Transgenic seeds harvested from CL4 and the screenhouse will be kept in hermetically-sealed aluminum foil packages and stored in locked refrigerators designated for storage of transgenic seeds in Rice Biotechnology Laboratory, lRRl. · Movement of all seeds and plant materials will be done in compliance of all relevant biosafety and phytosanitary requirements of the Philippines.

a) All activities shall be conducted at the Genetic Transformation Laboratory and CL4 Transgenic Greenhouse of the lnternational Rice Research lnstitute (lRRl) in Los Baños, Laguna.

b) The proponent shall adhere as closely as possible to the schedule of activities reflected in the submitted Gantt chart.

c) Any modifications in the schedule of activities shall be made with the concurrence of the lRRl-IBC and the DOST-BC.

d) A biosafety contingency plan shall be submitted before the conduct of the experiment.

e) The proponent shall inform which activities would require the presence of the IRRI-IBC, DOST-BC and PEQS-BPI personnel.

f) The proponent shall ensure that only the DOST-BC authorized personnel are allowed inside the experimental facilities.

g) The DOST-BC and PEQS-BPI personnel should be informed in advance of any visitations by unauthorized persons.

h) The DOST-BC and the PEQS-BPI personnel shall be informed immediately of any intrusion by unauthorized persons.

i) The proponent shall ensure that stray animals are excluded from the experimental facilities while tests are being conducted.

j) In case of undue destruction of experimental materials resulting from unauthorized entry of personnel or breach of containment of experimental facilities, the proponent shall implement measures to prevent the inadvertent escape of any viable material within the facility.

k) The proponent and the supervising IBC shall be held accountable for the undue destruction of the experimental materials and the consequences that their inadvertent escape may cause to the surrounding environment.

l) All viable materials within the experiment area shall be accounted for.

m) The proponent shall strictly observe proper disposal procedures for all materials.

n) Movement of all materials will be done in compliance with all relevant biosafety and phytosanitary requirements of the Philippines.

o) Any additional requirement that the DOST-BC may impose, as necessary, for the duration of the experiment shall be complied with.

p) The proponent shall submit through the IBC a completion report within 90 days after completion of the experiment.

q) The IBC shall submit to the DOST-BC a report on all completion of this project, in the prescribed format.

The world population reached a historic milestone of 7 billion recently and is expected to reach 9 billion by 2050. However, the decelerating rice yield growth in the last decade has been a matter of great concern for future global food security. To be able to produce enough rice to feed the growing global population, the world needs to use all available tools at its disposal, including GM technology, to improve productivity in a sustainable manner with fewer resources.

Precise and straight forward methods for editing plant genomes are needed for functional genomics and crop improvement. Targeted modification of plant genomes remains challenging due to ineffective methods for delivering reagents for genom engineering to plant cells. A vector having sequences mimicking plant Geminiviral sequence for delivery of the nuclease and the donor can enhance homologous recombination-mediated gene targeting at a reporter locus in rice. Other variations including replicons based on the bean yellow dward virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacteriums tumefaciencs T-DNA. A new set of DNA vectors will be imported and evaluated at IRRI from the University of Minnesota. All of these viral vectors are "disarmed", so they can't spread from cell to cell, at least not by infection. They can only replicate within the cell. The viral vectors are therefore not infectious anymore, because it cannot actively move from cell to cell - that's why we have to deliver it with Agrobacterium, and it will only replicate in cells where it was delivered by Agrobacterium.

Recent afvances in molecular engineering are focusing on endonucleases, which recognize unique sequences and specifically cleave DNA at unique sites within the genome. These nucleases have sequence recognition sites and a cleavage module/domain that can be used for gene targeting and editing. Most widely used sequence-specific nucleases include zinc finger nucleases (ZFNs), TAL (transcruiption activator-like) Effector Nucleases (TALENs) and the RNA-guided endonuclease Cas9 of the type II Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) nuclease (CRISPR-CAS) system. The latter have easy programmability using customizable short RNAs to target specific DNA sequences and brings unprecedented flexibility and versatility for targeted genome modification. We at IRRI along with the partner from Unicersity of Minnesota, pioneers in developing genome editing technology, propose to use innovative genome editing technologies that allow gene insertion at a single locus and generate complete gene knock out.

Main objective is to increase frequency of target modification in rice using Geminiviral vectors for delivery of the nucleases (ZFN, TALENs and CRISPR/CAS) and the donor to enhance homologous recombination-mediated gene targeting at a reporter locus in rice. To achieve this main objective we will: (i) transform site-specific integration donor plasmids constructs, containing the GFP marker gene, for the purpose of gene integration and mutation induction; (ii) develop the Nipponbare and/or Indica lines containing the TALENs and/or CRISPR/Cas platform at IRRI; (iii) test the vectors containing elements from Bean yellow dward virus, Tomato leaf curl and Wheat dwarf virus for effective delivery of genes in rice.