Abstract
CRISPR/Cas9 gene editing technology has successfully introduced modifications at target DNA sequences in many plant species including Arabidopsis. After the target gene is edited, the CRISPR/Cas9 construct needs to be removed to ensure genetic stability and to gain any regulatory approval for commercial applications. However, removal of the transgenes by genetic segregation, backcross, and genotyping is very laborious and time-consuming. The methods we report here allow fast and effective isolation of transgene-free T2 Arabidopsis plants with the desired modifications at the target genes. We express a fluorescence protein mCherry under the control of a seed-specific promoter At2S3 and placed the cassette into the CRISPR/Cas9 vector. Therefore, we can use mCherry as a proxy for the presence of Cas9, and we are able to visually isolate the Cas9-free Arabidopsis plants with heritable mutations at the T2 generation. We targeted two sites in the ABP1 gene to demonstrate the effectiveness of our approach.
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References
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. https://doi.org/10.1126/science.1231143
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826. https://doi.org/10.1126/science.1232033
Gao Y, Zhang Y, Zhang D, Dai X, Estelle M, Zhao Y (2015) Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development. Proc Natl Acad Sci U S A 112(7):2275–2280. https://doi.org/10.1073/pnas.1500365112
Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8(8):1274–1284. https://doi.org/10.1016/j.molp.2015.04.007
Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163(3):759–771. https://doi.org/10.1016/j.cell.2015.09.038
Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS (2016) Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34(8):863–868. https://doi.org/10.1038/nbt.3609
Sun Y, Zhang X, Wu C, He Y, Ma Y, Hou H, Guo X, Du W, Zhao Y, Xia L (2016) Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase. Mol Plant 9(4):628–631. https://doi.org/10.1016/j.molp.2016.01.001
He Y, Wang R, Dai X, Zhao Y (2017) On improving CRISPR for editing plant genes: ribozyme-mediated guide RNA production and fluorescence-based technology for isolating transgene-free mutants generated by CRISPR. Prog Mol Biol Transl Sci 149:151–166. https://doi.org/10.1016/bs.pmbts.2017.03.012
Tang X, Lowder LG, Zhang T, Malzahn AA, Zheng X, Voytas DF, Zhong Z, Chen Y, Ren Q, Li Q, Kirkland ER, Zhang Y, Qi Y (2017) A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat Plants 3:17103. https://doi.org/10.1038/nplants.2017.103
Yang N, Wang R, Zhao Y (2017) Revolutionize genetic studies and crop improvement with high-throughput and genome-scale CRISPR/Cas9 gene editing technology. Mol Plant 10(9):1141–1143. https://doi.org/10.1016/j.molp.2017.08.001
Gao X, Chen J, Dai X, Zhang D, Zhao Y (2016) An effective strategy for reliably isolating heritable and Cas9-free arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Plant Physiol 171(3):1794–1800. https://doi.org/10.1104/pp.16.00663
Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345. https://doi.org/10.1038/nmeth.1318
Gao Y, Zhao Y (2014) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol 56(4):343–349. https://doi.org/10.1111/jipb.12152
He Y, Zhang T, Yang N, Xu M, Yan L, Wang L, Wang R, Zhao Y (2017) Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing. J Genet Genomics 44(9):469–472. https://doi.org/10.1016/j.jgg.2017.08.003
Zhang T, Gao Y, Wang R, Zhao Y (2017) Production of guide RNAs in vitro and in vivo for CRISPR using ribozymes and RNA polymerase II promoters. Bio Protoc 7(4):e2148. https://doi.org/10.21769/BioProtoc.2148
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743
Acknowledgment
Our research is supported by the NIH grant R01GM114660 to YZ.
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Yu, H., Zhao, Y. (2019). Fluorescence Marker-Assisted Isolation of Cas9-Free and CRISPR-Edited Arabidopsis Plants. In: Qi, Y. (eds) Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8991-1_11
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DOI: https://doi.org/10.1007/978-1-4939-8991-1_11
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