DEVELOPMENT OF CAO-1 MUTANT IN RICE USING CRISPR/Cpf1 TECHNOLOGY
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Date
2020-10
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AAU, Jorhat
Abstract
CRISPR (Clustered Regularly Interspaced Short Pallindromic Repeats) is originally a bacterial immune system against virus based on RNA guided bacterial defence mechanisms designed to recognize and eliminate foreign DNA of invading bacteriophage and plasmid. Although the most successful genome editing tool used at present is CRISPR/Cas9 (Jaganathan et al., 2018), the newly developed CRISPR/cpf1 (CRISPR from Prevotella and Francisella 1) is a single RNA guided endonuclease of a class-II CRISPR-Cas system that has several advantages over CRISPR/Cas9 (Zetsche et al., 2015). Cpf1 needs single guiding RNA (sgRNA) while Cas9 requires two guide RNAs (crRNA and tracrRNA). While Cas9 make a blunt end cut of the DNA molecule after recognizing a PAM sequence NGG (Moon et al., 2018), the Cpf1 endonuclease makes a staggered cut of the DNA after recognizing a PAM sequence viz. NTTT, offering researchers more option when selecting an editing site. The current study employed CRISPR/Cpf1 construct pSS09 harbouring sgRNA of CAO1 gene of rice that was developed at Prof. Okita’s Lab, Institute of Biological Chemistry, Washington State University, Pullman, USA. The main objective of the present study was to validate the efficacy of CRISPR/Cpf1 system in rice. Chlorophyllide-a-oxygenase (CAO1) is an enzyme that converts chlorophyll a to chlorophyll b. Knockout of the gene using CRISPR technology is expected to produce “pale yellow” leaf phenotype. We introduced CRISPR/Cpf1 binary vector harbouring sgRNA of CAO1 gene into Japonica rice cv. Kitaake through Agrobacterium genetic transformation. Embryogenic calli were induced from mature seed with efficiency of 96.3% and regeneration efficiency of embryogenic calli was observed as 63.3%. 43 putative transformed lines were generated in the current study. Out of 43 transformed lines, 20 lines were selected based on varied level of pale green colour of the leaves and subjected to PCR analysis using gene specific primers for the targeted sequence of CAO1 gene. Transformed lines number 8, 9 and 13 showed mutations in the CAO1 gene with 9 bp, 12 bp and 25 bp deletions, respectively. We observed fairly high efficiency of mutation rate (2.22%) with CRISPR/Cpf1 system and the mutations occurred at very near the targeted sgRNA sequence of CAO1 gene.