Targeted editing of rice micro RNA osa-miR396b through CRISPR/Cas9 system (Record no. 290301)
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| 000 -LEADER | |
|---|---|
| fixed length control field | 06057nam a22001577a 4500 |
| 082 ## - DEWEY DECIMAL CLASSIFICATION NUMBER | |
| Classification number | 660.6 |
| Item number | SAN/TA PG |
| 100 ## - MAIN ENTRY--PERSONAL NAME | |
| Personal name | Sanjay Sathian |
| 245 ## - TITLE STATEMENT | |
| Title | Targeted editing of rice micro RNA osa-miR396b through CRISPR/Cas9 system |
| 260 ## - PUBLICATION, DISTRIBUTION, ETC. (IMPRINT) | |
| Place of publication, distribution, etc | Vellanikkara |
| Name of publisher, distributor, etc | Department of Plant Biotechnology, Centre for Plant Biotechnology and Molecular Biology, College of Agriculture |
| Date of publication, distribution, etc | 2022 |
| 300 ## - PHYSICAL DESCRIPTION | |
| Extent | 88p. |
| 502 ## - DISSERTATION NOTE | |
| Dissertation note | MSc |
| 520 ## - SUMMARY, ETC. | |
| Abstract | 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. |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Plant Biotechnology |
| 700 ## - ADDED ENTRY--PERSONAL NAME | |
| Personal name | Rehna Augustine (Guide) |
| 942 ## - ADDED ENTRY ELEMENTS (KOHA) | |
| Source of classification or shelving scheme | |
| Item type | Theses |
| Not for loan | Collection code | Permanent location | Current location | Shelving location | Date acquired | Full call number | Barcode | Date last seen | Koha item type |
|---|---|---|---|---|---|---|---|---|---|
| Not For Loan | Reference Book | KAU Central Library, Thrissur | KAU Central Library, Thrissur | Theses | 2023-05-12 | 660.6 SAN/TA PG | 175692 | 2023-05-12 | Theses |