Abstract
Key message
CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants.
Abstract
The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest. The present study utilized CRISPR/Cas9 and CRISPR-Cpf1 technology to knock out an early developmental gene EPFL9 (Epidermal Patterning Factor like-9, a positive regulator of stomatal development in Arabidopsis) orthologue in rice. Germ-line mutants that were generated showed edits that were carried forward into the T2 generation when Cas9-free homozygous mutants were obtained. The homozygous mutant plants showed more than an eightfold reduction in stomatal density on the abaxial leaf surface of the edited rice plants. Potential off-target analysis showed no significant off-target effects. This study also utilized the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to target the same OsEPFL9 gene to test the activity of this class-2 CRISPR system in rice and found that Cpf1 is also capable of genome editing and edits get transmitted through generations with similar phenotypic changes seen with CRISPR-Cas9. This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bae S, Park J, Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30(10):1473–1475. doi:10.1093/bioinformatics/btu048
Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42(22):e168–e168
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 system1. Plant Physiol 166:1292–1297
Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ (2013) Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res 23:465–472
Chen K, Gao C (2013) TALENs: customizable molecular DNA scissors for genome engineering of plants. J Genet Genomics 40:271–279
Cho SW, Kim S, Kim JM, Kim J-S (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232
Datta K, Datta SK (2006) Indica rice (Oryza sativa, BR29 and IR64). Methods Mol Biol 343:201–212
Deriano L, Roth DB (2013) Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage. Annu Rev Genet 47:433–455
Deskgen. http://www.deskgen.com
Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE (2014) Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nat Biotechnol 32:1262–1267
Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191. doi:10.1038/nbt.3437
Endo A, Masafumi M, Kaya H, Toki S (2016) Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Sci Rep 6:38169. doi:10.1038/srep38169
Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA (2013) Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods 10:741–743
Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–826
Hiei Y, Komari T (2008) Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3:824–834
Hill JT, Demarest BL, Bisgrove BW, Su YC, Smith M, Yost HJ (2014) Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products. Dev Dyn 243(12):1632–1636
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278
Hunt L, Bailey KJ, Gray JE (2010) The signalling peptide EPFL9 is a positive regulator of stomatal development. New Phytol 186:609–614
Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41:1–12
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier EA (2012) Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–21
Komor AC, Kim YB, Packer MS, Zuris J, Liu DR (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 61:5985–5991
Kondo T, Kajita R, Miyazaki A, Hokoyama M, Nakamura-Miura T, Mizuno S, Masuda Y, Irie K, Tanaka Y, Takada S, Kakimoto T (2010) Stomatal density is controlled by a mesophyll-derived signaling molecule. Plant Cell Physiol 51:1–8
Kumar V, Jain M (2015) The CRISPR-Cas system for plant genome editing: advances and opportunities. J Exp Bot 66:47–57
Lei Y, Lu L, Liu H-Y, Li S, Xing F, Chen L-L (2014) CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7:1494–1496
Li J, Meng X, Zong Y, Chen K, Zhang H, Liu J, Li J, Gao C (2016) Gene replacements and insertions in rice by intron targeting using CRISPR–Cas9. Nat Plants 2:16139
Liang Z, Zhang K, Chen K, Gao C (2014) Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. J Genet Genomics 41:63–68
Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM (2013) Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833–838
Polsky BS (2016) CRISPR-mediated base editing without DNA double-strand breaks. Mol Cell 62:477–478
Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK (2012) FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30:460–465
Rychel AL, Peterson KM, Torii KU (2010) Plant twitter: ligands under 140 amino acids enforcing stomatal patterning. J Plant Res 123(3):275–280
Saito K, Matsuda F (2010) Metabolomics for functional genomics, systems biology, and biotechnology. Annu Rev Plant Biol 61:463–489
Sauer NJ, Narváez-Vásquez J, Mozoruk J, Miller RB, Warburg ZJ, Woodward MJ, Mihiret YA, Lincoln TA, Segami RE, Sanders SL, Walker KA (2016) Oligonucleotide-mediated genome editing provides precision and function to engineered nucleases and antibiotics in plants. Plant Physiol 170(4):1917–1928
Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu JL, Gao C (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686–688
Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X (2013) Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 23:720–723
Sugano SS, Shimada T, Imai Y, Okawa K, Tamai A, Mori M, Hara-Nishimura I (2010) Stomagen positively regulates stomatal density in Arabidopsis. Nature 463:241–244
Takata N, Yokota K, Ohki S, Mori M, Taniguchi T, Kurita M (2013) Evolutionary relationship and structural characterization of the EPF/EPFL gene family. PLoS One 8(6):e65183. doi:10.1371/journal.pone.0065183
van Campen J, Yaapar MN, Narawatthana S, Lehmeier C, Wanchana S, Thakur V, Chater C, Kelly S, Rolfe SA, Quick WP, Fleming AJ (2016) Combined chlorophyll fluorescence and transcriptomic analysis identifies the P3/P4 transition as a key stage in rice leaf photosynthetic development. Plant Physiol. doi:10.1104/pp.15.01624
Voytas DF (2013) Plant genome engineering with sequence-specific nucleases. Annu Rev Plant Biol 64:327–350
Xie K and Yang Y (2013) RNA-Guided genome editing in plants using a CRISPR-Cas system. Mol Plant 6:1975–1983
Xie K, Zhang J, Yang Y (2014) Genome-wide prediction of highly specific guide RNA spacers for the CRISPR–Cas9-Mediated genome editing in model plants and major crops. Mol Plant 1:1–10
Xu RF, Li H, Qin RY, Li J, Qiu CH, Yang YC, Ma H, Li L, Wei PC, Yang JB (2015) Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Sci Rep 5:11491
Xu R, Qin R, Li H, Li D, Li L, Wei P, Yang J (2016) Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnol J. doi:10.1111/pbi.12669
Yan M, Zhou SR, Xue HW (2015) CRISPR Primer Designer: Design primers for knockout and chromosome imaging CRISPR‐Cas system. J Integr Plant Biol 57(7):613–617
Yoshida R, Saito MM, Nagao H, Higuchi T (2010) Bayesian experts in exploring reaction kinetics of transcription circuits. Bioinformatics 26:i589–i595
Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163.3: 759–771
Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, Qiu JL, Gao C (2016) Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun, vol 7. doi:10.1038/ncomms12617
Acknowledgements
We thank Prof. Caixia Gao of Chinese Academy of Science, China for providing us Cas9 and gRNA scaffold constructs. Also we would like to thank Dr. Feng Zhang of Broad Institute MIT for providing us pcDNA3-huLbCpf1. We also thank Florencia Montecillo and Juvy Reyes for helping in rice transformation and molecular work respectively.
Author contribution statement
XY, AKB and JD contributed equally. AB, PQ, JG and XY designed the experiments and wrote the manuscript. Vector designing and construction was done by XY, AKB, AB and TK (CRISPR-508 Cas9). XY,SM, CPB, CC, AB (CRISPR-Cpf1). Surveyor assay and sequencing was performed 509 by KP, XY, SM, CPB (Cas9 plants and Cpf1 plants) and AKB (Cas9 plants). Rice 510 transformation was supervised by XY. Microscopy was performed by JD, HL and RC. Data 511 analysis was performed by AB and XY. Southern blotting was performed by CPB.
Author information
Authors and Affiliations
Contributions
AB, PQ, JG and XY designed the experiments and wrote the manuscript. Vector designing and construction was done by XY, AKB, AB and TK (CRISPR-Cas9). XY,SM, CPB, CC, AB (CRISPR-Cpf1). Surveyor assay and sequencing was performed by KP, XY, SM, CPB (Cas9 plants and Cpf1 plants) and AKB (Cas9 plants). Rice transformation was supervised by XY. Microscopy was performed by JD, HL and RC. Data analysis was performed by AB and XY. Southern blotting was performed by CPB.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing financial interests.
Additional information
Communicated by Nese Sreenivasulu.
Xiaojia Yin, Akshaya K. Biswal and Jacqueline Dionora have Contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Yin, X., Biswal, A.K., Dionora, J. et al. CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice. Plant Cell Rep 36, 745–757 (2017). https://doi.org/10.1007/s00299-017-2118-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-017-2118-z