Are Non-Target Mutations via CRISPR in Plants a Concern?

I provide outreach on GE crops for the University of Kentucky, so I am very attentive to questions of risks and benefits regarding CRISPR-based genetic modification. I occasionally have heard or read expressions of concern regarding the capacity of CRISPR/Cas9 to induce genetic change in DNA sequences that are highly similar—but not identical—to sequences that are the target of the designed single-guide RNA. These are often referred to as “non-target mutations.

While I initially considered this to be a potentially important issue, over time and through study and reflection, I have come to seriously question the notion that this represents a significant concern. Here are my reasons:

1. In plants, reported off-target rates are commonly low to non-existent [1-13];

2. Strategies for reducing off-target mutations continue to be researched and published [9, 13-20];

3. Off-target mutations can be monitored via whole-genome sequencing;

4. In sexually reproducing crops, undesirable mutations can be segregated out [15];

5. In contrast to clinical applications in humans, the relevance of non-target genetic changes during crop improvement is questionable. Clearly, eliminating non-target genetic changes during human gene therapy would be highly desirable. The same may be said for gene drive-modified organisms [21]. However, in the case of crop plants, I am aware of no evidence suggesting that non-target genetic changes in a crop improvement program present an intrinsic risk to biosafety or to human health. Indeed, more genetic, transcriptomic, and proteomic change is commonly observed from conventional breeding that from GE [22-35]; therefore, one could postulate greater off-target risks from conventional breeding than from CRISPR-based technologies. (My intention is not to raise anxieties over the safety of either general approach to crop improvement. Genetic crop improvement has one of the safest records of all human endeavors. My intention is merely to put genetic changes associated with CRISPR in perspective.)

Literature Cited
1. Peterson, B. A., Haak, D. C., Nishimura, M. T., Teixeira, P. J., James, S. R., Dangl, J. L. and Nimchuk, Z. L., Genome-Wide Assessment of Efficiency and Specificity in CRISPR/Cas9 Mediated Multiple Site Targeting in Arabidopsis. PLoS One, 2016, Vol. 11, p. e0162169, DOI: 10.1371/journal.pone.0162169. Available from: https://www.ncbi.nlm.nih.gov/pubmed/27622539
2. Woo, J. W., Kim, J., Kwon, S. I., Corvalan, C., Cho, S. W., Kim, H., Kim, S. G., Kim, S. T., Choe, S. and Kim, J. S., DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol, 2015, Vol. 33, p. 1162-4, DOI: 10.1038/nbt.3389. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26479191
3. Yang, H., Wu, J. J., Tang, T., Liu, K. D. and Dai, C., CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus. Sci Rep, 2017, Vol. 7, p. 7489, DOI: 10.1038/s41598-017-07871-9. Available from: https://www.ncbi.nlm.nih.gov/pubmed/28790350
4. Zhang, Y., Liang, Z., Zong, Y., Wang, Y., Liu, J., Chen, K., Qiu, J. L. and Gao, C., Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun, 2016, Vol. 7, p. 12617, DOI: 10.1038/ncomms12617. Available from: https://www.ncbi.nlm.nih.gov/pubmed/27558837
5. Zhang, H., Zhang, J., Wei, P., Zhang, B., Gou, F., Feng, Z., Mao, Y., Yang, L., Zhang, H., Xu, N. and Zhu, J. K., The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J, 2014, Vol. 12, p. 797-807, DOI: 10.1111/pbi.12200. Available from: https://www.ncbi.nlm.nih.gov/pubmed/24854982
6. Wang, P., Zhang, J., Sun, L., Ma, Y., Xu, J., Liang, S., Deng, J., Tan, J., Zhang, Q., Tu, L., Daniell, H., Jin, S. and Zhang, X., High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system. Plant Biotechnol J, 2018, Vol. 16, p. 137-150, DOI: 10.1111/pbi.12755. Available from: https://www.ncbi.nlm.nih.gov/pubmed/28499063
7. Sanchez-Leon, S., Gil-Humanes, J., Ozuna, C. V., Gimenez, M. J., Sousa, C., Voytas, D. F. and Barro, F., Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9. Plant Biotechnol J, 2018, Vol. 16, p. 902-910, DOI: 10.1111/pbi.12837. Available from: https://www.ncbi.nlm.nih.gov/pubmed/28921815
8. Macovei, A., Sevilla, N. R., Cantos, C., Jonson, G., Slamet-Loedin, I., Cermak, T., Voytas, D., Choi, I. R. and Chadha-Mohanty, P., Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted mutagenesis confer resistance to Rice tungro spherical virus. Plant Biotechnol J, 2018, DOI: 10.1111/pbi.12927. Available from: https://www.ncbi.nlm.nih.gov/pubmed/29604159
9. Belhaj, K., Chaparro-Garcia, A., Kamoun, S. and Nekrasov, V., Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods, 2013, Vol. 9, p. 39, DOI: 10.1186/1746-4811-9-39. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24112467
10. Ueta, R., Abe, C., Watanabe, T., Sugano, S. S., Ishihara, R., Ezura, H., Osakabe, Y. and Osakabe, K., Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9. Sci Rep, 2017, Vol. 7, p. 507, DOI: 10.1038/s41598-017-00501-4. Available from: https://www.ncbi.nlm.nih.gov/pubmed/28360425
11. Nekrasov, V., Wang, C., Win, J., Lanz, C., Weigel, D. and Kamoun, S., Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Sci Rep, 2017, Vol. 7, p. 482, DOI: 10.1038/s41598-017-00578-x. Available from: https://www.ncbi.nlm.nih.gov/pubmed/28352080
12. Fister, A. S., Landherr, L., Maximova, S. N. and Guiltinan, M. J., Transient Expression of CRISPR/Cas9 Machinery Targeting TcNPR3 Enhances Defense Response in Theobroma cacao. Front Plant Sci, 2018, Vol. 9, p. 268, DOI: 10.3389/fpls.2018.00268. Available from: https://www.ncbi.nlm.nih.gov/pubmed/29552023
13. Scheben, A., Wolter, F., Batley, J., Puchta, H. and Edwards, D., Towards CRISPR/Cas crops – bringing together genomics and genome editing. New Phytol, 2017, Vol. 216, p. 682-698, DOI: 10.1111/nph.14702. Available from: https://www.ncbi.nlm.nih.gov/pubmed/28762506
14. Paul, J. W., 3rd and Qi, Y., CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects. Plant Cell Rep, 2016, Vol. 35, p. 1417-27, DOI: 10.1007/s00299-016-1985-z. Available from: https://www.ncbi.nlm.nih.gov/pubmed/27114166
15. Yin, K., Gao, C. and Qiu, J. L., Progress and prospects in plant genome editing. Nat Plants, 2017, Vol. 3, p. 17107, DOI: 10.1038/nplants.2017.107. Available from: https://www.ncbi.nlm.nih.gov/pubmed/28758991
16. Schiml, S., Fauser, F. and Puchta, H., The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J, 2014, Vol. 80, p. 1139-50, DOI: 10.1111/tpj.12704. Available from: https://www.ncbi.nlm.nih.gov/pubmed/25327456
17. Bortesi, L. and Fischer, R., The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv, 2015, Vol. 33, p. 41-52, DOI: 10.1016/j.biotechadv.2014.12.006. Available from: https://www.ncbi.nlm.nih.gov/pubmed/25536441
18. Chen, J. S., Dagdas, Y. S., Kleinstiver, B. P., Welch, M. M., Sousa, A. A., Harrington, L. B., Sternberg, S. H., Joung, J. K., Yildiz, A. and Doudna, J. A., Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature, 2017, Vol. 550, p. 407-410, DOI: 10.1038/nature24268. Available from: https://www.ncbi.nlm.nih.gov/pubmed/28931002
19. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. and Joung, J. K., Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol, 2014, Vol. 32, p. 279-284, DOI: 10.1038/nbt.2808. Available from: https://www.ncbi.nlm.nih.gov/pubmed/24463574
20. Hu, J. H., Miller, S. M., Geurts, M. H., Tang, W., Chen, L., Sun, N., Zeina, C. M., Gao, X., Rees, H. A., Lin, Z. and Liu, D. R., Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 2018, Vol. 556, p. 57-63, DOI: 10.1038/nature26155. Available from: https://www.ncbi.nlm.nih.gov/pubmed/29512652
21. Hayes, K. R., Hosack, G. R., Dana, G. V., Foster, S. D., Ford, J. H., Thresher, R., Ickowicz, A., Peel, D., Tizard, M., De Barro, P., Strive, T. and Dambacher, J. M., Identifying and detecting potentially adverse ecological outcomes associated with the release of gene-drive modified organisms. Journal of Responsible Innovation, 2018, Vol. 5, p. S139-S158, DOI: 10.1080/23299460.2017.1415585. Available
22. Ricroch, A. E., Assessment of GE food safety using ‘-omics’ techniques and long-term animal feeding studies. N Biotechnol, 2013, Vol. 30, p. 349-54, DOI: 10.1016/j.nbt.2012.12.001. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23253614
23. Schnell, J., Steele, M., Bean, J., Neuspiel, M., Girard, C., Dormann, N., Pearson, C., Savoie, A., Bourbonniere, L. and Macdonald, P., A comparative analysis of insertional effects in genetically engineered plants: considerations for pre-market assessments. Transgenic Res, 2015, Vol. 24, p. 1-17, DOI: 10.1007/s11248-014-9843-7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25344849
24. Gao, L., Cao, Y., Xia, Z., Jiang, G., Liu, G., Zhang, W. and Zhai, W., Do transgenesis and marker-assisted backcross breeding produce substantially equivalent plants? A comparative study of transgenic and backcross rice carrying bacterial blight resistant gene Xa21. BMC Genomics, 2013, Vol. 14, p. 738, DOI: 10.1186/1471-2164-14-738. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24165682
25. Batista, R., Saibo, N., Lourenco, T. and Oliveira, M. M., Microarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion. Proc Natl Acad Sci U S A, 2008, Vol. 105, p. 3640-5, DOI: 10.1073/pnas.0707881105. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18303117
26. Lehesranta, S. J., Davies, H. V., Shepherd, L. V., Nunan, N., McNicol, J. W., Auriola, S., Koistinen, K. M., Suomalainen, S., Kokko, H. I. and Karenlampi, S. O., Comparison of tuber proteomes of potato varieties, landraces, and genetically modified lines. Plant Physiol, 2005, Vol. 138, p. 1690-9, DOI: 10.1104/pp.105.060152. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15951487
27. Ladics, G. S., Bartholomaeus, A., Bregitzer, P., Doerrer, N. G., Gray, A., Holzhauser, T., Jordan, M., Keese, P., Kok, E., Macdonald, P., Parrott, W., Privalle, L., Raybould, A., Rhee, S. Y., Rice, E., Romeis, J., Vaughn, J., Wal, J. M. and Glenn, K., Genetic basis and detection of unintended effects in genetically modified crop plants. Transgenic Res, 2015, Vol. 24, p. 587-603, DOI: 10.1007/s11248-015-9867-7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25716164
28. El Ouakfaoui, S. and Miki, B., The stability of the Arabidopsis transcriptome in transgenic plants expressing the marker genes nptII and uidA. Plant J, 2005, Vol. 41, p. 791-800, DOI: 10.1111/j.1365-313X.2005.02350.x. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15743445
29. Herman, R. A. and Price, W. D., Unintended compositional changes in genetically modified (GM) crops: 20 years of research. J Agric Food Chem, 2013, Vol. 61, p. 11695-701, DOI: 10.1021/jf400135r. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23414177
30. Herman, R. A., Fast, B. J., Scherer, P. N., Brune, A. M., de Cerqueira, D. T., Schafer, B. W., Ekmay, R. D., Harrigan, G. G. and Bradfisch, G. A., Stacking transgenic event DAS-O15O7-1 alters maize composition less than traditional breeding. Plant Biotechnol J, 2017, Vol. 15, p. 1264-1272, DOI: 10.1111/pbi.12713. Available from: https://www.ncbi.nlm.nih.gov/pubmed/28218975
31. Harrigan, G. G., Venkatesh, T. V., Leibman, M., Blankenship, J., Perez, T., Halls, S., Chassy, A. W., Fiehn, O., Xu, Y. and Goodacre, R., Evaluation of metabolomics profiles of grain from maize hybrids derived from near-isogenic GM positive and negative segregant inbreds demonstrates that observed differences cannot be attributed unequivocally to the GM trait. Metabolomics, 2016, Vol. 12, p. 82, DOI: 10.1007/s11306-016-1017-6. Available from: https://www.ncbi.nlm.nih.gov/pubmed/27453709
32. Wang, Y. M., Dong, Z. Y., Zhang, Z. J., Lin, X. Y., Shen, Y., Zhou, D. and Liu, B., Extensive de Novo genomic variation in rice induced by introgression from wild rice (Zizania latifolia Griseb.). Genetics, 2005, Vol. 170, p. 1945-56, DOI: 10.1534/genetics.105.040964. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15937131
33. Weber, N., Halpin, C., Hannah, L. C., Jez, J. M., Kough, J. and Parrott, W., Editor’s choice: Crop genome plasticity and its relevance to food and feed safety of genetically engineered breeding stacks. Plant Physiol, 2012, Vol. 160, p. 1842-53, DOI: 10.1104/pp.112.204271. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23060369
34. Baudo, M. M., Lyons, R., Powers, S., Pastori, G. M., Edwards, K. J., Holdsworth, M. J. and Shewry, P. R., Transgenesis has less impact on the transcriptome of wheat grain than conventional breeding. Plant Biotechnol J, 2006, Vol. 4, p. 369-80, DOI: 10.1111/j.1467-7652.2006.00193.x. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17177803
35. Anderson, J. E., Michno, J. M., Kono, T. J., Stec, A. O., Campbell, B. W., Curtin, S. J. and Stupar, R. M., Genomic variation and DNA repair associated with soybean transgenesis: a comparison to cultivars and mutagenized plants. BMC Biotechnol, 2016, Vol. 16, p. 41, DOI: 10.1186/s12896-016-0271-z. Available from: https://www.ncbi.nlm.nih.gov/pubmed/27176220

Advertisements