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Molecular Breeding

, 36:108 | Cite as

CRISPR/Cas9 activity in the rice OsBEIIb gene does not induce off-target effects in the closely related paralog OsBEIIa

  • Can Baysal
  • Luisa Bortesi
  • Changfu Zhu
  • Gemma Farré
  • Stefan Schillberg
  • Paul Christou
Article

Abstract

Genome editing with the CRISPR/Cas9 system allows mutations to be induced at any 20-bp target site in the genome preceded by the short protospacer adjacent motif (PAM) 5′-NGG-3′. The brevity and degeneracy of the PAM ensures that the motif occurs every ~10 bp in plant genomes, and all plant genes therefore contain many targetable sites. However, the CRISPR/Cas9 system tolerates up to three mismatches in the target site, so the ability to target genes in a specific manner requires the design of synthetic guide RNAs (sgRNAs) that do not bind off-target sites anywhere else in the genome. This is straightforward for single-copy genes but more challenging if a target gene has one or more paralogs because the principles that balance targeting efficiency (the frequency of on-target mutations) and accuracy (the absence of off-target mutations) are not fully understood and may be partially species-dependent. To investigate this phenomenon in rice, we targeted the rice starch branching enzyme IIb gene (OsBEIIb) with two sgRNAs designed to differ at two and six positions, respectively, from corresponding sites in the close paralog OsBEIIa. In each case, half of the mismatches were in the essential seed region immediately upstream of the PAM, where exact pairing is thought to be necessary, and the other half were in the distal part of the target. The sgRNAs also differed in predicted targeting efficiency (39 and 96 %, respectively). We found that the sgRNA with the low predicted efficiency was actually the most efficient in practice, achieving a mutation frequency of 5 % at the target site, whereas the sgRNA with the high predicted efficiency generated no mutations at the second target site. Furthermore, neither of the sgRNAs induced an off-target mutation in the OsBEIIa gene. Our data indicate that efficiency predictions should be tested empirically because they do not always reflect the experimental outcome and that a 1-bp mismatch in the seed region of a sgRNA is sufficient to avoid off-target effects even in closely related rice genes.

Keywords

Genome editing Isozyme Mutation frequency Off-target activity Oryza sativa Starch branching enzyme Targeted mutation 

Notes

Acknowledgments

We would like to thank Dr. Caixia Gao (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) for providing pJIT163-2NLSCas9 containing the cas9 gene codon optimized for rice and the empty pU3-gRNA vector for the introduction of sgRNAs into rice. Synthetic biology and genome editing work at the UdL is supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2014-54426-P and BIO2014-54441-P) and the Catalan Autonomous Government 2014 SGR 1296 Agricultural Biotechnology Research Group.

Authors’ contributions

CB, LB, GF, CZ, SS and PC formulated the problem and designed the research. CB carried out the experiments. All co-authors discussed results and formulated conclusions. CB and PC wrote the paper.

Supplementary material

11032_2016_533_MOESM1_ESM.docx (16 kb)
Supplementary material 1 (DOCX 15 kb)
11032_2016_533_MOESM2_ESM.docx (18 kb)
Supplementary material 2 (DOCX 17 kb)
11032_2016_533_MOESM3_ESM.docx (510 kb)
Supplementary Figure 1 Sanger sequencing results representing independent transgenic lines. (A) Sequence traces with multiple peaks at the target site are shown before cloning (left) with multiple peaks, and after cloning (right) without multiple peaks. (B) Sequence trace of wild-type rice callus DNA for comparison (DOCX 509 kb)

References

  1. Baltes NJ, Voytas DF (2015) Enabling plant synthetic biology through genome engineering. Trends Biotechnol 33:120–131CrossRefPubMedGoogle Scholar
  2. Bassie L, Zhu C, Romagosa I, Christou P, Capell T (2008) Transgenic wheat plants expressing an oat arginine decarboxylase cDNA exhibit increases in polyamine content in vegetative tissue and seeds. Mol Breed 22:39–50CrossRefGoogle Scholar
  3. Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9:39–49CrossRefPubMedPubMedCentralGoogle Scholar
  4. Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V (2015) Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol 32:76–84CrossRefPubMedGoogle Scholar
  5. Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52CrossRefPubMedGoogle Scholar
  6. Brinkman EK, Chen T, Amendola M, Van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucl Acids Res 42:e168CrossRefPubMedPubMedCentralGoogle Scholar
  7. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:756–761CrossRefGoogle Scholar
  8. Dian W, Jiang H, Wu P (2005) Evolution and expression analysis of starch synthase III and IV in rice. J Exp Bot 56:623–632CrossRefPubMedGoogle Scholar
  9. Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346:1077–1086CrossRefGoogle Scholar
  10. Endo M, Mikami M, Toki S (2015) Multigene knockout utilizing off-target mutations of the CRISPR/cas9 system in rice. Plant Cell Physiol 56:41–47CrossRefPubMedGoogle Scholar
  11. Farré G, Sudhakar D, Naqvi S, Sandmann G, Christou P, Capell T, Zhu C (2012) Transgenic rice grains expressing a heterologous ρ-hydroxyphenylpyruvate dioxygenase shift tocopherol synthesis from the γ to the α isoform without increasing absolute tocopherol levels. Transgenic Res 21:1093–1097CrossRefPubMedGoogle Scholar
  12. 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 31:822–826CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marrafini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832CrossRefPubMedPubMedCentralGoogle Scholar
  14. Jinek M, Chylinski K, Fonfara I, Hauer M, Charpentier E, Doudna JA (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefPubMedGoogle Scholar
  15. Kang T-J, Yang M-S (2004) Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants. BMC Biotechnol 4:20CrossRefPubMedPubMedCentralGoogle Scholar
  16. Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain. Proc Natl Acad Sci USA 93:1156–1160CrossRefPubMedPubMedCentralGoogle Scholar
  17. Konieczny A, Ausubel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J 4:403–410CrossRefPubMedGoogle Scholar
  18. Li J, Zhang B, Ren Y, Gu S, Xiang Y, Huang C, Du J (2015) Intron targeting-mediated and endogenous gene integrity-maintaining knockin in zebrafish using the CRISPR/Cas9 system. Cell Res 25:634–637CrossRefPubMedPubMedCentralGoogle Scholar
  19. Li M, Li X, Zhou Z, Wu P, Fang M, Pan X, Lin Q, Luo W, Wu G, Li H (2016) Reassessment of the four yield-related genes Gn1a, IPA1, DEP1 and GS3 in rice using a CRISPR/cas9 system. Front Plant Sci 7:377PubMedPubMedCentralGoogle Scholar
  20. Liang G, Zhang H, Lou D, Yu D (2016) Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing. Sci Rep 6:21451CrossRefPubMedPubMedCentralGoogle Scholar
  21. Liu L, Fan X-D (2014) CRISPR–Cas system: a powerful tool for genome engineering. Plant Mol Biol 85:209–218CrossRefPubMedGoogle Scholar
  22. Ma X, Zhang Q, Zhu Q, Liu W, ChenY Qiu R, Wang B, Yang Z, Li H, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu Y (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284CrossRefPubMedGoogle Scholar
  23. Milbury CA, Li J, Makrigiorgos GM (2011) Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations. Nucl Acids Res 39:1–10CrossRefGoogle Scholar
  24. Mizuno K, Kobayashi E, Tachibana M, Tachibana M, Kawasaki T, Fujimura T, Funane K, Kobayashi M, Baba T (2001) Characterization of an isoform of rice starch branching enzyme, RBE4, in developing seeds. Plant Cell Physiol 42:349–357CrossRefPubMedGoogle Scholar
  25. Nakamura Y (2002) Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue. Plant Cell Physiol 43:718–725CrossRefPubMedGoogle Scholar
  26. Nishi A, Nakamura Y, Tanaka N, Satoh H (2001) Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm. Plant Physiol 127:459–472CrossRefPubMedPubMedCentralGoogle Scholar
  27. Osakabe Y, Osakabe K (2015) Genome editing with engineered nucleases in plants. Plant Cell Physiol 56:389–400CrossRefPubMedGoogle Scholar
  28. Rahman S, Regina A, Li Z, Mukai Y, Yamamoto M, Kosar-Hasemi B, Abrahams S, Morell MK (2015) Comparison of starch-branching enzyme genes reveals evolutionary relationships among isoforms. characterization of a gene for starch-branching enzyme IIa from the wheat D genome donor Aegilops tauschii 1. Plant Physiol 125:1314–1324CrossRefGoogle Scholar
  29. Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, Van Der Ost J, Brouns SJJ, Severinov K (2011) Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci USA 108:10098–10103CrossRefPubMedPubMedCentralGoogle Scholar
  30. Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi J, Qiu J, Gao C (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686–688CrossRefPubMedGoogle Scholar
  31. Smith AM, Denyer K, Martin C (1999) The synthesis of the starch granule. Plant Physiol 48:67–87Google Scholar
  32. Sudhakar D, Duc LT, Bong BB, Tinjuangjun P, Maqbool SB, Valdez M, Jefferson R, Christou P (1998) An efficient rice transformation system utilizing mature seed-derived explants and a portable, inexpensive particle bombardment device. Transgenic Res 7:289–294CrossRefGoogle Scholar
  33. Valdez M, Cabrera-Ponce JL, Sudhakar D, Herrera-Estrella L, Christou P (1998) Transgenic Central American, West African and Asian Elite rice varieties resulting from particle bombardment of foreign DNA into mature seed-derived explants utilizing three different bombardment devices. Ann Bot 82:795–801CrossRefGoogle Scholar
  34. Weeks DP, Spalding MH, Yang B (2016) Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnol J 14:483–495CrossRefPubMedGoogle Scholar
  35. Xie K, Yang Y (2013) RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant 6:1975–1983CrossRefPubMedGoogle Scholar
  36. Xie K, Zhang J, Yang Y (2014) Genome-wide prediction of highly specific guide RNA spacers for CRISPR-Cas9-mediated genome editing in model plants and major crops. Mol Plant 7:923–926CrossRefPubMedGoogle Scholar
  37. Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N, Zhu JK (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Department of Plant Production and Forestry Science, School of Agrifood and Forestry Science and Engineering (ETSEA)University of Lleida-Agrotecnio CenterLleidaSpain
  2. 2.Institute for Molecular BiotechnologyRWTH Aachen UniversityAachenGermany
  3. 3.Fraunhofer Institute for Molecular Biology and Applied Ecology IMEAachenGermany
  4. 4.ICREA, Catalan Institute for Research and Advanced StudiesBarcelonaSpain

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