2.1. Plant Germination

In our laboratory, we cultivated the superior Indica rice cultivar RP Bio-226. All transgenic plants as well as wild-type RP Bio-226 plants were grown in the fields at Hyderabad under typical growing circumstances in a greenhouse between 28°C and 35°C. Rice callus was grown in a regular nutritional medium for 25 days following germination on a 96-well plate to test for blast resistance at the callus stage [18]. They were put in a growth chamber with 70% relative humidity for 10 h at 20°C during the night time and 14 h at approximately 30°C during the day time under natural sunlight.

2.2. Construction of CRISPR Vector

The sgRNA expression vector (pRGEB31gRNA) and the plant expression gene cassette with Cas9 (pRGEB31CRISPR/Cas9) were purchased from Addgene company [19]. A suitable target sequence was chosen 20 bases upstream region of the protospacer adjacent motif (PAM), in accordance with the design guidelines of the CRISPR/CASs technique [20]. A BLAST scan of the sequences, including PAM, against the rice genome was conducted to establish the targeted sequences’ targeting specificity in the rice genome. The target region containing gRNA-forward/gRNA-reverse primers was synthesized by the Bioserve Company (https://bioserve.in/) and both primers were hybridized by annealing [Table 1].

Table 1: Primer sequence of sgRNA (forward and reverse) with restriction sites.

Following that, the sgRNA-expressing cassette pRGEB31sgRNA was cloned with the target region having chimeric primers using a BsaI site [21]. The hybridized molecule is now called an insert. In comparison with the non-targeted region in the PAM or PAM-proximal area, the targeted specific sequence differs by one base. Through overlapping polymerase chain reaction (PCR), the sgRNA-expression cassette was created. The 5′ ends of the primers were inserted with the sgRNA’s target-specific sequence (target OsHDT701).

2.3. Development of Recombinant Vector

The total length of the CRISPR vector is 15 kb whereas the size of the designed sgRNA is 32 base pairs. Both the CRISPR vector and the sgRNA were subjected to restriction digestion with the BSA1 restriction enzyme. Once the plasmid and the sgRNA were properly digested with the enzyme, a ligation reaction was set up with the vector and insert DNA. The transformation was carried out and the ligated product was then transferred to the top 10 Escherichia coli competent cells through the heat shock method. The plasmid was isolated and run on an electrophoresis unit and a PCR was set as initial denaturation – 94°C for 5 min, cyclic denaturation – 94°C for 30 s, annealing –56°C for 30 s, elongation – 72°C for 30 s, and final elongation – 72°C for 10 min with the universal M13 forward and M13 reverse primers for the recombinant plasmid confirmation [Table 2].

Table 2: Primer used for PCR analysis of recombinant bacterial colonies.

2.4. Rice Genetic Transformation with Agrobacterium

Using the heat shock method, the Cas9/sgRNA-expressing binary vector PRGEB31 was introduced into the Agrobacterium tumefaciens strain LBA 4404. It was done to transform the embryogenic calli from the RP Bio-226 Indica rice variety using an Agrobacterium. In a nutshell, kanamycin-resistant calli were selected using the kanamycin-containing medium. After that, the selected calli were placed on regeneration media to help transgenic plants grow [15]. After 60–90 days of culture, the transgenic seedlings are transplanted onto a grassland area in the course of the rice harvesting period.

2.5. Identification of Mutated Transgenic Plants

To find the mutation at the desired location, genomic DNA was extracted from the transgenic plants’ leaves using the CTAB procedure. Utilizing PCR mastermix, the isolated DNA with a concentration of 25 ng was employed as the template to conduct PCR. With the help of a particular pair of designed primers that surround the intended target position, PCR was carried out with the initial denaturation –94°C for 5 min, cyclic denaturation – 94°C for 30 s, annealing – 62°C for 30 s, elongation – 72°C for 30 s, and final elongation – 72°C for 10 min for amplifying the genomic area that contains the CRISPR-Cas9 target site. The amplified product was directly sequenced using the Sanger sequencing method. The sequence obtained from both the transgenic as well as the wild-type plants was compared to find out the mutations. The term “homozygote mutation” was used to describe mutations with typical sequencing chromatograms.

With the aid of agarose gel electrophoresis and PCR analysis, plants were separated into T-DNA-free and Cas9-free plants. RPBio-226 DNA as a (+) control and sterile distilled H2O as a (−) control was employed, together with the pRGEB31CRISPR/Cas9 plasmids and the transgenic plants. T-DNA-free plants were those that tested negative for Cas9.

2.6. Green House Trial for Blast Resistance

A blast test was conducted to assess the blast disease in plants at the seedling stage. In the greenhouse, we assessed the blast resistance of 4-week-old wild type and mutated homozygous plants and these 4 weeks plants were subjected to freshwater treatment. Analyzing the plant height, leaf size, leaf shape, leaf length, panicle length, and the leaf response to the blast resistance was measured.

3.1. CRISPR/Cas9 Designing

A 20 basepair nucleotide sequence in OsHDT701’s exon number one was selected as the mutation’s targeted region to create a sudden gene change that surely targets OsHDT701 gene sequence in RPBio-226 cv. of rice [Figure 1a]. The gene’s coding region had a predicted Cas9 cleavage site that was 28 bp downside of the start point (ATG) [Figure 2]. The twofold plasmid called Cas9-OsHDT701-gRNA [Figure 1b] was created with the help of the CRISPR-Cas9 gene cassette reported by Ma et al. [22]. Through Agrobacterium-mediated transformation, the vector was employed to modify the rice variety RPBio-226. The plant DNA was isolated and amplified by the DNA that contains the targeted region to assess the effectiveness of OsHDT701 in gene editing. Six kanamycin-resistant transgenic RPBio-226 plants yielded RPBio-226 mutants that could be identified using site-specific PCR analysis and the Sanger sequencing method. Sequencing revealed that there could be a deletion of a single nucleotide.

Figure 1: OsHDT701 gene alteration was caused through CRISPR-Cas9 in the RP Bio-226 rice variety. (a) Structural representation of OsHDT701 gene targeting sequence. (b) Structural representation of transfer-DNA region of the CRISPR-Cas9 vector pRGEB31 targeting construct along with the sgRNA targeting OsHDT701 gene. [Click here to view]

Figure 2: The target gene’s specifics. (a) Structural representation of the OsHDT gene and the targeted position on the 1st exon to develop sgRNA. [Click here to view]

The homozygous mutant lines had shown one base pair deletion at the 174^th position in the OsHDT701 gene. Allele mutation caused by the OsHDT701 gene was observed in the mutant lines, but the transferred DNA was not present. As a result of this finding, it can be demonstrated that CRISPR/Cas9 has the ability to edit the rice blast gene to produce a mutant rice plan.

3.2. Confirmation of Recombinant Clone

The recombinant plasmid was isolated and when run on the 1.0% agarose gel electrophoresis yielded more than a 15 kb fragment (vector 15kb + insert 32 bp) [Figure 3]. This implies that both the vector as well as the insert have properly ligated along with the restriction sites in the recombinant clone [Figure 4]. A PCR reaction was set up with M13 forward and M13 reverse primers to double-check and confirm the recombinant clone on 1% agarose gel [Figure 5].

Figure 3: Confirmation of plasmid DNA and insert. Lane-1: 1 kb DNA ladder; Lane-λ: Hind III λ DNA marker; Lane-1: Plasmid DNA; Lane-2: Insert DNA. [Click here to view]

Figure 4: Restriction digestion of plasmid and recombinant plasmid DNA on 3.5% gel. Lane-1: 1 kb DNA ladder; Lane-λ: Hind III λ DNA marker; Lane-1,2: Plasmid DNA; Lane-3,4: recombinant plasmid DNA. [Click here to view]

Figure 5: Polymerase chain reaction (PCR) confirmation of recombinant plasmid. Lane-1: 50bp DNA ladder; Lane-1,2,3,4: PCR confirmation of insert DNA (32 base pairs). [Click here to view]

3.3. Selection of Stable Homozygous and CAS Free Edited Lines

We created the sequence targeting primers Cas9p-forward and Cas9p-reverse [Table 3] and conducted PCR for mutant plants to evaluate the feasibility of generating rice lines retaining the necessary alterations in OsHDT701 gene without the T-DNA of the vector pRGEB31 [Figure 2a]. Cas9 PCR results that were negative indicated that there were no transgenes present. A majority of mutant plants obtained are T-DNA-free plants. These findings demonstrated that there is a possibility of obtaining a T-DNA-free homozygous mutant population. To identify the traits associated with blast resistance, we identified homozygous T-DNA-free mutant lines and named them RPBio-226 cas-1, RPBio-226 cas-2, RPBio-226 cas-3, and RPBio-226 cas-4. This finding suggests that the plants without transfer-DNA and with the necessary target sequence changes will be obtained by CRISPR/Cas9-induced gene mutations in the OsHDT701 gene, thus enhancing blast resistance in rice plants.

Table 3: Primer sequence of Cas 9 (forward and reverse).

Developing desirable mutant phenotypes with traditional breeding methods is a difficult exercise that requires producing and screening hundreds of gene-mutated offspring [23]. Gene edition is a regulated target-specific procedure that enables DNA edition with molecular tools, such as ZFNs, TALENs, and CRISPR/Cas. In genome editing technology the binding of proteins and enzymes is different for ZFNs, Talens, and CRISPR. DNA binding protein and the enzyme Fok1 bind ZFNs and TALENS whereas the Cas9 enzyme binds CRISPR forming a CRISPR/Cas9 system. This CRISPR/Cas9 system is a RNA-dependent nuclease system. The whole method of CRISPR depends on the RNA sequence and this helps to guide the breakdown of nuclease enzyme. CRISPR nucleases and Cas9 nucleases are the two components of this system. A CRISPR nuclease sequence consists of repeats that are <50 base pairs in length and are separated by distinct spacers of comparable length. However, Cas9 nuclease is the CRISPR-associated protein that promotes the events of degrading the foreign DNA [24]. The foreign DNA is identified during the pathogen attack and the sequence is incorporated into the CRISPR sequence. A type of bacterial genome impression is developed as a result of this. The CRISPR sequence recognizes the foreign DNA and degrades with the assistance of the Cas9 enzyme [25] when attacked for the 2^nd time. This entire DNA degradation process was assisted by the Cas9 enzyme. Given its capacity to cause double-strand breaks in the genome, the CRISPR-Cas9 system is a fantastic tool for use as genomic scissors during gene editing. The preparation of the CRISPR/Cas9 system in the laboratory requires.

The double-stranded CRISPR breaks can be repaired either using a low-fidelity non-homologs end joining (NHEJ) method or a more specialized homology-directed repair system [27]. Because NHEJ is error-prone, it can alter the target sequence by inserting or deleting (InDels), which causes a loss of function and results in genetically modified plants. Transformation, screening, and molecular characterization of the desirable trait will be done once the PAM sequence is identified and the guide RNA is synthesized [28].

A CRISPR/Cas9 mechanism developed with a Cas9 endonuclease and an RNA guide complex has shown significantly improved gene editing efficiency [20]. Although TALENs and CRISPR-Cas9 are both useful technologies for gene alteration, they each have unique benefits and drawbacks. CRISPR-Cas9 expressing gene constructs are substantially simpler in construction which takes just 2–3 days for completion when compared to TALENs which usually take 7–10 days for construction [29]. In addition, compared to TALENs, CRISPR/Cas9 causes a substantially greater incidence of mutations, the frequency of the mutation that was targeted using CRISPR-Cas9 ranges from a percentage of 21–66 for 11 rice genes [30]. Similarly, Ma et al. described that editing 46 target regions in the rice genome using CRISPR-Cas9 had an average mutational rate of 85%. Corn, wheat, rice, and soybeans [31] are just a few of the key crops that have benefited from the widespread usage of CRISPR/Cas9 technology thus far.

3.4. RPBio-226 rice Mutants Showed Increased Resistance to M. oryzae

Homozygous mutant lines were exposed to the fungal microorganism M. oryzae strain B157 during the sapling stage to identify the resistance toward the blast trait of the mutant plants in rice. Due to its highest pathogenic nature the isolate B157 being extremely powerful resisted the infection whereas the wild plants being extremely vulnerable, the leaves were almost dried with the fungal disease [Figure 6] [32]. However, in comparison to wild-type plants, all mutant rice lines had much fewer places where the pathogen had caused lesion formation. By quantifying the lesion regions, the differences were further assessed and it was shown the mutated plants had an improved resistance to the blast disease. At the tillering stage, pathogen-induced lesion lengths were likewise shorter in mutant rice lines than in wild-type plants, and all gene-mutated plants had significant differences from the non-mutated wild plants, according to the lesion length formed. This finding suggests a deletion mutation in the OsHDT701 gene increases M. oryzae resistance in mutated rice plants and that implies the OsHDT701 gene adversely controls rice’s blast resistance.

Figure 6: Identification of blast resistance in wild-type and cas-free edited homozygous mutant rice lines (RPBio-226 cas-1, RPBio-226 cas-2, RPBio-226 cas-3, and RPBio-226 cas-4). [Click here to view]

3.5. The Major Agronomic Features were Unaffected in Induced RPBio-226 Mutant Rice

We examined visually the plant height, leaf size, shape, length, panicle length, and leaf response of all homozygous mutant lines [Figure 6] to ascertain if mutations in the OsHDT701 gene had an impact on agronomic variables. In terms of the agronomic traits examined, we hardly found any prominent difference when both the homozygous mutated plants and wild plants were compared under normal growing conditions.