MSc

Rice (Oryza sativa L.) is one of the most produced and consumed food crops in
the world. There is an urgent need to increase rice production to feed the increasing
population. Rice yield is determined by several components like grain size, grain
weight, number and architecture of panicles, number of spikelets per panicle and grain
filling. The microRNA family osa-miR396 is known to suppress the expression of rice
growth regulating factors (OsGRFs) resulting in reduced growth and yield. The miRNA
osa-miR396b is reported to be a negative regulator of spikelet number and inflorescence
development. CRISPR/Cas9 mediated knockout of osa-miR396b gene can thus
possibly result in an enhanced yield in rice. Hence, the current study ‘Targeted editing
of rice microRNA, osa-miR396b through CRISPR/Cas9 system’ was conducted during
the period from 2019 to 2022 at the Department of Plant Biotechnology, College of
Agriculture, Kerala Agricultural University, Vellanikkara, Thrissur.
Oryza sativa ssp. japonica cultivar Nipponbare was selected for the study due
to well established transformation protocols and higher transformation efficiency.
Initially, the sequence information of the rice microRNA gene osa-miR396b was
retrieved from ‘miRbase’. The stem loop sequence obtained was used to design guide
RNAs (gRNAs) using the software CRISPR-P v2.0 and CRISPR-PLANT v2. The
gRNAs were selected for further studies mainly based on GC content and number of
off-target sites. The target osa-miR396b gene sequence was confirmed by amplifying
the genomic region flanking the target using gene specific primers followed by
sequencing and the sequence analysis using Clustal Omega and BLASTn showed 100%
similarity with reported sequences.
The osa-miR396b G1 CRISPR/Cas9 construct was generated by restriction
digestion of CRISPR/Cas9 binary vector pRGEB32 using BsaI restriction enzyme
followed by ligation with annealed and phosphorylated gRNA. The osa-miR396b G1
construct was cloned to E. coli strain DH5α. The positive clones were confirmed by
Sanger sequencing of the plasmid DNA isolated from the colonies and sequence
analysis using Clustal Omega. Three (osa-miR396b G1 #2, osa-miR396b G1 #3 and
osa-miR396b G1 #4) out of four plasmids sequenced were having gRNA insertion. The
osa-miR396b G1 #4 CRISPR/Cas9 construct was mobilized to A. tumefaciens strain
EHA105. Positive clones were confirmed by PCR amplification of hygromycin
resistance gene (hptII) using specific primers. Colony #1 of A. tumefaciens with osamiR396b G1 CRISPR/Cas9 construct out of the two positive colonies was used for rice
transformation.
Genetic transformation of rice was achieved through Agrobacterium-mediated
transformation. The first step was induction of calli from dehusked and sterilized
Nipponbare seeds. Five-day-old calli were infected with Agrobacterium harbouring
osa-miR396b G1 CRISPR/Cas9 construct for 1.5-2 min. and co-cultivated for 48 hours.
An empty vector was also transformed as control. After washing off excess
Agrobacterium growth with sterile distilled water containing Augmentin (300 mgL-1
)
or carbenicillin (250 mgL-1
), the calli were kept for selection in selection medium
supplemented with hygromycin (50 mgL-1
) and Augmentin (300 mgL-1
) or carbenicillin
(400 mgL-1
). The calli showing proliferation of microcalli were transferred to
regeneration medium supplemented with NAA (0.02 mgL-1
) and kinetin (2.0 mgL-1
)
for inducing somatic embryogenesis. The somatic embryos were allowed to develop
into small plantlets which were transferred to rooting medium for root development.
The rooted plantlets were initially maintained in sterile distilled water and then
hardened in sterile soil-cocopeat mixture in paper cups and transferred to pots with soilsand-cow dung mixture. A total of 94 putative transformed plants for osa-miR396b G1
CRISPR/Cas9 construct and four vector control plants were obtained. For confirming
successful transformation, PCR amplification of hptII gene using hygromycin gene
specific primers was done. DNA extracted from 35 plants and two vector control were
used as PCR template. A total of 16 out of 35 transformed and two vector control plants
were hygromycin positive, indicating successful transformation.
The osa-mir396b partial gene sequence was amplified using gene specific
primers and sequenced by Sanger sequencing for detecting mutation. Detection of
mutation was carried out using ‘Inference of CRISPR Edits (ICE)’ software by
Synthego. Analysis using ‘ICE’ detected indel mutations in seven plants. Five plants
(71.42%) had deletions and two (28.57%) had insertions around the cut site. Four plants
(57.14%) had heterozygous mutations (mutation in one allele) and three (42.86%) had
chimeric (more than two) mutations. The mutation efficiency was calculated to be
43.75%. The mutations obtained could lead to a non-functional osa-miR396b gene in
these plants. The study successfully demonstrated application of CRISPR/Cas9 system
to mutate rice microRNA gene. The knockout of osa-miR396b gene will likely promote
the expression of rice GRF genes improving the grain yield. Further studies should be
conducted to study inheritance pattern of mutations in the subsequent generations.
Genotypic and phenotypic analyses is to be done to study effect of mutated osamiR396b gene on its target genes and on yield and other agronomically important traits.