TY  - BOOK
AU  - Sanjay Sathian
AU  - Rehna Augustine (Guide)
TI  - Targeted editing of rice micro RNA osa-miR396b through CRISPR/Cas9 system
U1  - 660.6 
PY  - 2022///
CY  - Vellanikkara
PB  - Department of Plant Biotechnology, Centre for Plant Biotechnology and Molecular Biology, College of Agriculture
KW  - Plant Biotechnology
N1  - MSc
N2  - 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
ER  -