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Rice (Oryza sativa L.) is the most widely consumed staple food of world’s
human population belonging to Asia and Africa. Being a semi-aquatic annual plant, rice
is highly prone to losses due to various environmental stresses. Many studies regarding
this had revealed the need for developing varieties tolerant to abiotic and biotic stresses.
Various methods like Marker Assisted Breeding, mutation breeding, RNAi, Antisense
technology, ZFNs and TALENs were in use to develop elite traits for abiotic stress
tolerance in crops like rice. But very recently, CRISPR/Cas9 system had come into the
limelight as an efficient tool for the genetic manipulations of crops. Studies have
identified OsMADS26 transcription factor as a negative regulator of drought tolerance
in rice. Hence the current study, ‘Editing of rice transcription factor OsMADS26 for
drought tolerance through CRISPR/Cas9 system’ was undertaken during the period
from 2019 to 2021 at the Centre for Plant Biotechnology and Molecular Biology, CoA,
Vellanikkara, Thrissur with an objective to develop drought tolerance in rice.
The rice cultivar Nipponbare was selected for the study due to its competence
in genetic transformation and regeneration. For CRISPR/Cas9 mediated targeted
editing of OsMADS26 gene, guide RNAs (gRNAs) were designed using online software
CRISPR-P v2. Genome sequence information of OsMADS26 gene available from rice
genome annotation project was used for the study. Genomic region of OsMADS26
gene, flanking the gRNA target (~ 450 bp) was amplified using gene specific primers
and sequence of the target region was confirmed using BLASTn and ClustalW analysis.
The CRISPR/Cas9 binary vector pRGEB32 was used to clone the guide RNAs using
BsaI restriction sites. Three gRNAs were selected for cloning based on features like on
score value (higher the value better the editing efficiency), GC content, (40-60%), no.
of off-target sites (Minimum number of off-target sites preferred), presence of
secondary structure, location on the genome (towards 5' end of gene in exonic region is
preferred) etc.
The CRISPR/Cas9 construct for cloning was developed by annealing and
ligating the gRNAs to the pRGEB32 vector followed by cloning in E. coli strain DH5α.
The putative positive clones were identified by colony PCR and further confirmed by
Sanger sequencing. The plasmids isolated from PCR positive colonies were sequenced
using universal M13 Reverse primer which is present on pRGEB32 vector. The
sequences of the clones were confirmed using multiple sequence alignment tool
ClustalW. One colony of gRNA 1 construct (OsMADS26 #G1-1) and two colonies of
gRNA 3 (OsMADS26 #G3-3 and OsMADS26 #G3-4) were found positive.
The CRISPR/Cas9 constructs of OsMADS26 were then mobilized into
Agrobacterium tumefaciens strain EHA105 following the Freeze-thaw method. The
positive clones were identified using plasmid PCR using hygromycin gene specific
primers. Positive colonies of OsMADS26 #G1-1 and OsMADS26 #G3-3 constructs in
EHA105 were then used for rice genetic transformation.
The seeds of Oryza sativa sub species japonica cultivar Nipponbare were
inoculated into N6 medium supplemented with 3.0 mgL-1 2,4-D for callus induction.
After five days, the calli were infected with Agrobacterium cultures harboring desired
gRNA constructs for 1.5-2 min. Along with the gRNA constructs, an empty vector was
also transformed to rice as vector control and a set of untransformed culture were also
maintained. After around two days of co-cultivation, the excess Agrobacterium growth
was washed-off thoroughly from the calli using the bacteriostatic agent Augmentin.
The calli were then placed on selection medium containing Augmentin and
Hygromycin. The hygromycin resistant calli showed proliferation after 14 days of
incubation. The proliferating microcalli were then transferred to regeneration medium
after 21 days. Proliferation of microcalli was observed in vector control, wild type as
well as OsMADS26 #G1-1 and OsMADS26 #G3-3 co-transformed plates. The vector
control and untransformed calli showed greening and shoot primordia initiation in
regeneration medium. The regenerated shoots will be analyzed for mutation in future.
Hence, in the current study, gRNA constructs for targeted editing of OsMADS26 gene
was successfully developed and transformed in to rice cultivar Nipponbare. Rice
genetic transformation suitable to our lab conditions were also optimized. Rice plants
with mutations in the OsMADS26 gene is expected in future which can confer drought
tolerance.