Dual-targeting by CRISPR/Cas9 for precise excision of transgenes from rice genome
- 1.2k Downloads
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system has emerged as the robust gene editing tool that functions through the double-stranded break repair process leading to targeted mutagenesis in higher genomes. CRISPR/Cas9 has been simplified to a two component system consisting of a single guide RNA (gRNA) that binds Cas9 to target genomic sites in sequence-dependent manner. This RNA-guided nuclease system has mostly been applied for inducing point mutations or short insertion-deletions at one or multiple loci. The present study addressed the utility of this system for excising marker genes from plant genomes, an application highly relevant for developing marker-free transgenic plants. A transgenic rice line expressing β-glucuronidase (GUS) gene was transformed by Agrobacterium or gene gun with a construct expressing Cas9 and two gRNAs to target each end of 1.6 kb GUS gene. Molecular analysis of the transformed lines detected excision at low frequency in the callus lines, but at significantly higher frequency in plant lines, indicating robust efficiency of Cas9:gRNA in regenerated plants. Bi-allelic excisions were observed in plants derived from three independent events, allowing recovery of homozygous excision lines in the first generation (T0). Notably, the excision in different plant lines was formed by precise cut and ligation of the two blunt ends without mutation at or around the excision site. Since the goal of marker-removal technologies is to precisely excise a defined piece of DNA without introducing mutations in the adjacent sequences, Cas9:gRNA system could be an effective tool for producing marker-free plants.
KeywordsCRISPR/Cas9 gRNA Multiplex gene editing Marker excision Rice transformation
The vectors pRGEB32, pRGE32 and pGTR were donated by Yinong Yang and obtained from Addgene.com. This project is supported by the Arkansas Division of Agriculture.
JLU developed vectors and performed a part of rice transformations. SZ did most of the rice transformations and PCR analysis. VS analyzed the data and wrote the manuscript.
- Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166:1292–1297. doi: 10.1104/pp.114.247577 CrossRefPubMedPubMedCentralGoogle Scholar
- Feng ZY, Mao YF, Xu NF, Zhang BT, Wei PL, Wang Z, Zhang ZJ, Yang DL, Yang L, Zeng L, Liu XD, Zhu J-K (2014) Multi-generation analysis reveals the inheritance, specificity and patterns of CRISPR/Cas induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632–4637. doi: 10.1073/pnas.1400822111 CrossRefPubMedPubMedCentralGoogle Scholar
- Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31:688–691. doi: 10.1038/nbt.2654 CrossRefPubMedPubMedCentralGoogle Scholar
- Luo K, Duan H, Zhao D, Zheng X, Deng X, Chen Y, Stewart CN Jr, McAvoy R, Wu Y, Jiang X, He A, Pei Y, Li Y (2007) ‘GM-gene-deletor’: fusedloxP-FRTrecognition sequences dramatically improve efficiency of FLP or Cre recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol J 5:263–374. doi: 10.1111/j.1467-7652.2006.00237.x CrossRefPubMedGoogle Scholar
- Srivastava V, Akbudak MA, Nandy S (2011) Marker-free plant transformation. In: Dan Y, Ow DW (eds) Historical technology developments in plant transformation. Bentham eBooks, pp 108–122. doi: 10.2174/97816080524861110101