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 | 06126nam a22001697a 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 <br/>the world. There is an urgent need to increase rice production to feed the increasing<br/>population. Rice yield is determined by several components like grain size, grain <br/>weight, number and architecture of panicles, number of spikelets per panicle and grain <br/>filling. The microRNA family osa-miR396 is known to suppress the expression of rice <br/>growth regulating factors (OsGRFs) resulting in reduced growth and yield. The miRNA <br/>osa-miR396b is reported to be a negative regulator of spikelet number and inflorescence <br/>development. CRISPR/Cas9 mediated knockout of osa-miR396b gene can thus <br/>possibly result in an enhanced yield in rice. Hence, the current study ‘Targeted editing <br/>of rice microRNA, osa-miR396b through CRISPR/Cas9 system’ was conducted during <br/>the period from 2019 to 2022 at the Department of Plant Biotechnology, College of <br/>Agriculture, Kerala Agricultural University, Vellanikkara, Thrissur.<br/>Oryza sativa ssp. japonica cultivar Nipponbare was selected for the study due <br/>to well established transformation protocols and higher transformation efficiency. <br/>Initially, the sequence information of the rice microRNA gene osa-miR396b was <br/>retrieved from ‘miRbase’. The stem loop sequence obtained was used to design guide <br/>RNAs (gRNAs) using the software CRISPR-P v2.0 and CRISPR-PLANT v2. The <br/>gRNAs were selected for further studies mainly based on GC content and number of <br/>off-target sites. The target osa-miR396b gene sequence was confirmed by amplifying <br/>the genomic region flanking the target using gene specific primers followed by <br/>sequencing and the sequence analysis using Clustal Omega and BLASTn showed 100% <br/>similarity with reported sequences.<br/>The osa-miR396b G1 CRISPR/Cas9 construct was generated by restriction <br/>digestion of CRISPR/Cas9 binary vector pRGEB32 using BsaI restriction enzyme <br/>followed by ligation with annealed and phosphorylated gRNA. The osa-miR396b G1 <br/>construct was cloned to E. coli strain DH5α. The positive clones were confirmed by <br/>Sanger sequencing of the plasmid DNA isolated from the colonies and sequence <br/>analysis using Clustal Omega. Three (osa-miR396b G1 #2, osa-miR396b G1 #3 and <br/>osa-miR396b G1 #4) out of four plasmids sequenced were having gRNA insertion. The <br/>osa-miR396b G1 #4 CRISPR/Cas9 construct was mobilized to A. tumefaciens strain <br/>EHA105. Positive clones were confirmed by PCR amplification of hygromycin <br/>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 <br/>transformation.<br/>Genetic transformation of rice was achieved through Agrobacterium-mediated <br/>transformation. The first step was induction of calli from dehusked and sterilized<br/>Nipponbare seeds. Five-day-old calli were infected with Agrobacterium harbouring<br/>osa-miR396b G1 CRISPR/Cas9 construct for 1.5-2 min. and co-cultivated for 48 hours.<br/>An empty vector was also transformed as control. After washing off excess <br/>Agrobacterium growth with sterile distilled water containing Augmentin (300 mgL-1<br/>) <br/>or carbenicillin (250 mgL-1<br/>), the calli were kept for selection in selection medium <br/>supplemented with hygromycin (50 mgL-1<br/>) and Augmentin (300 mgL-1<br/>) or carbenicillin <br/>(400 mgL-1<br/>). The calli showing proliferation of microcalli were transferred to <br/>regeneration medium supplemented with NAA (0.02 mgL-1<br/>) and kinetin (2.0 mgL-1<br/>)<br/>for inducing somatic embryogenesis. The somatic embryos were allowed to develop <br/>into small plantlets which were transferred to rooting medium for root development. <br/>The rooted plantlets were initially maintained in sterile distilled water and then <br/>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 <br/>CRISPR/Cas9 construct and four vector control plants were obtained. For confirming <br/>successful transformation, PCR amplification of hptII gene using hygromycin gene <br/>specific primers was done. DNA extracted from 35 plants and two vector control were <br/>used as PCR template. A total of 16 out of 35 transformed and two vector control plants <br/>were hygromycin positive, indicating successful transformation.<br/>The osa-mir396b partial gene sequence was amplified using gene specific <br/>primers and sequenced by Sanger sequencing for detecting mutation. Detection of <br/>mutation was carried out using ‘Inference of CRISPR Edits (ICE)’ software by <br/>Synthego. Analysis using ‘ICE’ detected indel mutations in seven plants. Five plants <br/>(71.42%) had deletions and two (28.57%) had insertions around the cut site. Four plants <br/>(57.14%) had heterozygous mutations (mutation in one allele) and three (42.86%) had <br/>chimeric (more than two) mutations. The mutation efficiency was calculated to be <br/>43.75%. The mutations obtained could lead to a non-functional osa-miR396b gene in <br/>these plants. The study successfully demonstrated application of CRISPR/Cas9 system <br/>to mutate rice microRNA gene. The knockout of osa-miR396b gene will likely promote <br/>the expression of rice GRF genes improving the grain yield. Further studies should be <br/>conducted to study inheritance pattern of mutations in the subsequent generations. <br/>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) |
| 856 ## - ELECTRONIC LOCATION AND ACCESS | |
| Uniform Resource Identifier | https://krishikosh.egranth.ac.in/handle/1/5810225375 |
| 942 ## - ADDED ENTRY ELEMENTS (KOHA) | |
| Source of classification or shelving scheme | Dewey Decimal Classification |
| Item type | Theses |
| Not for loan | Collection code | Home library | Current library | 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 | 12/05/2023 | 660.6 SAN/TA PG | 175692 | 12/05/2023 | Theses |