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BioDesign Research / 2020 / Article

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Volume 2020 |Article ID 8659064 |

Yingxiao Zhang, Yiping Qi, "Diverse Systems for Efficient Sequence Insertion and Replacement in Precise Plant Genome Editing", BioDesign Research, vol. 2020, Article ID 8659064, 4 pages, 2020.

Diverse Systems for Efficient Sequence Insertion and Replacement in Precise Plant Genome Editing

Received23 Jun 2020
Accepted19 Jul 2020
Published28 Jul 2020


CRISPR-mediated genome editing has been widely applied in plants to make uncomplicated genomic modifications including gene knockout and base changes. However, the introduction of many genetic variants related to valuable agronomic traits requires complex and precise DNA changes. Different CRISPR systems have been developed to achieve efficient sequence insertion and replacement but with limited success. A recent study has significantly improved NHEJ- and HDR-mediated sequence insertion and replacement using chemically modified donor templates. Together with other newly developed precise editing systems, such as prime editing and CRISPR-associated transposases, these technologies will provide new avenues to further the plant genome editing field.

1. Main Text

Clustered Regularly Interspaced Short Palindromic Repeats- (CRISPR-) mediated genome editing is a powerful and versatile tool for manipulating nucleic acids. However, targeted sequence insertion and replacement remains a significant challenge in plants. The efficiency of homology-directed repair (HDR) is usually low and inconsistent; as in plant somatic tissues, the predominant pathway to repair double-strand breaks (DSBs) is nonhomologous end joining (NHEJ). Many attempts have been made to improve HDR efficiencies, including actively selecting desired sequences (such as selective markers) [13], manipulating repair pathways [4], enriching donor templates near Cas nucleases [5], and increasing the amount of donor templates by simply providing more template or by using novel strategies such as geminivirus replication [68] and RNA transcription [9]. NHEJ-mediated gene insertion has also been demonstrated in plants with low editing efficiencies [10]. To develop a robust and efficient method to achieve targeted sequence insertion and replacement in plants, Lu et al. has improved the NHEJ-mediated DNA insertion approach using chemically modified donor DNA (Figure 1(a)) [11]. Furthermore, this approach has been leveraged to develop a tandem repeat-facilitated HDR strategy (TR-HDR) (Figure 1(b)) [11]. This study significantly increased the efficiencies of NHEJ- and HDR-mediated DNA insertion and replacement when compared to previous studies. The demonstrated methodology should be applicable in other plants and enable precise genome editing in basic plant research and crop breeding.

Lu et al. first focused on NHEJ-mediated gene insertion. To improve the double-stranded DNA (dsDNA) donor stability, two phosphorothioate linkages were added at the 5 and 3 ends of both DNA strands. In addition, 5-phosphorylation has been used to facilitate the NHEJ repair [11, 12]. Significantly higher insertion efficiencies were observed using modified dsDNA compared to unmodified dsDNA and single-stranded DNA (ssDNA). When targeting multiple genes separately or simultaneously using short (<70 bp) modified dsDNA donors, high efficiencies (10.6%-47.3%) were observed, suggesting this approach is highly robust and efficient. Furthermore, longer donors with lengths of 526 bp and 2,049 bp were simultaneously inserted into two loci, with 25.5% and 10.5% combined (two loci) insertion frequencies, respectively, indicating this approach is capable of inserting long DNA fragments.

Lu et al. further applied this approach to improve HDR efficiency. When tandemly repeated sequences are present near DSBs, higher HDR efficiencies have been observed. This is likely due to the repeat sequences being used as a repair template, based on the synthesis-dependent strand annealing (SDSA) mechanism [13]. Therefore, Lu et al. developed a tandem repeat-HDR strategy (TR-HDR) to achieve targeted sequence replacement. A repeat sequence with desired edits is inserted to serve as a template, using the firstly established insertion strategy. At the same time, a target site for the same guide RNA (gRNA) is formed between the two repeats, thus inducing the DSB followed by HDR. This method was successfully used to introduce base substitutions and in-locus tags. The precise editing efficiencies ranged from 3.4 to 11.4%. TR-HDR provided a novel avenue to obtain robust sequence replacement and insertion through HDR.

Collectively, this study made a breakthrough for targeted sequence insertion and replacement in plants that are amenable to biolistic delivery. High editing efficiencies were achieved through NHEJ (25% on average) and TR-HDR (6.1% on average), without actively selecting edited sequences, allowing complex, precise editing to be efficiently achieved at any desired genomic locus. This simple and reliable strategy was demonstrated to introduce gene regulatory elements, protein tags, and multiple base changes. It could be further applied for gene insertion and replacement, multiplexed single nucleotide polymorphism (SNP) introduction, promoter engineering, etc.

It is notable that this technology can be further improved by overcoming some limitations discussed in this study. The first concern is the random insertion of the repair template and CRISPR reagents into the plant genome, which is also the common problem caused by biolistic delivery. Although this issue can be resolved by generating large populations followed by selection, it would be time- and labor-consuming in practice. Using chemically modified ssDNA as the donor is another option to minimize off-target insertions (Figure 1(c)). Although unmodified ssDNA performed poorly in this study, editing efficiency could be significantly increased when using chemically modified templates. In addition, ribonucleoprotein (RNP) delivery of CRISPR reagents has been successfully used for plant genome editing to achieve transgene-free genome editing and limit off-target effects [14, 15] (Figure 1(c)). However, RNP-mediated sequence insertion and replacement is still a substantial challenge in plants. The second concern is the chimerism in plants, likely because edits happened at late stages of the transgenic plant regeneration. One possible solution is to further improve the editing efficiency. Localized donor enrichment through protein-protein or protein-DNA interactions can be considered [5, 1619] (Figure 1(c)). Moreover, Cas12-induced DSBs with staggered ends could potentially facilitate donor insertion (Figure 1(c)).

Recently, CRISPR technologies for sequence insertion and replacement have been advanced rapidly. Prime editing utilizes a reverse transcriptase fused to a Cas9-H840 nickase and a prime editing guide RNA (pegRNA) that encodes the target sequence and the desired edit, to achieve substitutions, deletions, and up to 44 bp insertions [20] (Figure 1(d)). Since prime editing enables flexible genome edits without introducing DSBs and donor templates, it has been applied in plants albeit with low efficiencies in many cases [2126]. To achieve large sequence insertion, a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) has been characterized [27] (Figure 1(e)). ShCAST, consisting of Cas12k and three Tn7-like transposase subunits, is able to integrate DNA into specific Escherichia coli genomic sites 60 to 66 bp downstream of the protospacer adjacent motif (PAM) with up to 80% efficiency [27]. Although this type of DNA insertion is not seamless, it can still be used to insert sequences to introns, around regulatory elements, and in safe harbor loci. However, this system has not been demonstrated in any other organisms. These technologies, along with the one developed by Lu et al., will expand the scope and capabilities of precise and complex genome editing in plants, leading to more biotechnological and agricultural applications.


The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of these funding agencies.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Authors’ Contributions

YZ and YQ participated in the inception, writing, and revising of the manuscript.


This work was supported by the National Science Foundation Plant Genome Research Program grants (award nos. IOS-1758745 and IOS-2029889), Biotechnology Risk Assessment Grant Program competitive grant (award no. 2018-33522-28789) from the US Department of Agriculture, Syngenta, and Foundation for Food and Agriculture Research grant (award no. 593603).


  1. Y. Sun, X. Zhang, C. Wu et al., “Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase,” Molecular Plant, vol. 9, no. 4, pp. 628–631, 2016. View at: Publisher Site | Google Scholar
  2. S. Li, J. Li, J. Zhang et al., “Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice,” Journal of Experimental Botany, vol. 69, no. 20, pp. 4715–4721, 2018. View at: Publisher Site | Google Scholar
  3. S. Li, Y. Zhang, L. Xia, and Y. Qi, “CRISPR-Cas12a enables efficient biallelic gene targeting in rice,” Plant Biotechnology Journal, vol. 18, no. 6, pp. 1351–1353, 2020. View at: Publisher Site | Google Scholar
  4. Y. Qi, Y. Zhang, F. Zhang et al., “Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways,” Genome Research, vol. 23, no. 3, pp. 547–554, 2013. View at: Publisher Site | Google Scholar
  5. Z. Ali, A. Shami, K. Sedeek et al., “Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology- directed repair for precise genome engineering in rice,” Communications Biology, vol. 3, no. 1, p. 44, 2020. View at: Publisher Site | Google Scholar
  6. M. Wang, Y. Lu, J. R. Botella, Y. Mao, K. Hua, and J. Zhu, “Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR/Cas9 system,” Molecular Plant, vol. 10, no. 7, pp. 1007–1010, 2017. View at: Publisher Site | Google Scholar
  7. T. Dahan-Meir, S. Filler-Hayut, C. Melamed-Bessudo et al., “Efficientin plantagene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system,” The Plant Journal, vol. 95, no. 1, pp. 5–16, 2018. View at: Publisher Site | Google Scholar
  8. Q. Shan, N. J. Baltes, P. Atkins et al., “ZFN, TALEN and CRISPR-Cas9 mediated homology directed gene insertion in _Arabidopsis_ : A disconnect between somatic and germinal cells,” Journal of Genetics and Genomics, vol. 45, no. 12, pp. 681–684, 2018. View at: Publisher Site | Google Scholar
  9. S. Li, J. Li, Y. He et al., “Precise gene replacement in rice by RNA transcript-templated homologous recombination,” Nature Biotechnology, vol. 37, no. 4, pp. 445–450, 2019. View at: Publisher Site | Google Scholar
  10. J. Li, X. Meng, Y. Zong et al., “Gene replacements and insertions in rice by intron targeting using CRISPR- Cas9,” Nature Plants, vol. 2, no. 10, p. 16139, 2016. View at: Publisher Site | Google Scholar
  11. Y. Lu, Y. Tian, R. Shen et al., “Targeted, efficient sequence insertion and replacement in rice,” Nature Biotechnology, pp. 1–6, 2020. View at: Publisher Site | Google Scholar
  12. S. Q. Tsai, Z. Zheng, N. T. Nguyen et al., “GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases,” Nature Biotechnology, vol. 33, no. 2, pp. 187–197, 2015. View at: Publisher Site | Google Scholar
  13. J. Steinert, S. Schiml, and H. Puchta, “Homology-based double-strand break-induced genome engineering in plants,” Plant Cell Reports, vol. 35, no. 7, pp. 1429–1438, 2016. View at: Publisher Site | Google Scholar
  14. Z. Liang, K. Chen, T. Li et al., “Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes,” Nature Communications, vol. 8, no. 1, p. 14261, 2017. View at: Publisher Site | Google Scholar
  15. S. Svitashev, C. Schwartz, B. Lenderts, J. K. Young, and A. M. Cigan, “Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes,” Nature Communications, vol. 7, no. 1, p. 13274, 2016. View at: Publisher Site | Google Scholar
  16. K. S. Ghanta, G. A. Dokshin, A. Mir et al., “5’ Modifications improve potency and efficacy of DNA donors for precision genome editing,” in bioRxiv, no. article 354480, 2018. View at: Publisher Site | Google Scholar
  17. J. Carlson-Stevermer, A. A. Abdeen, L. Kohlenberg et al., “Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing,” Nature Communications, vol. 8, no. 1, p. 1711, 2017. View at: Publisher Site | Google Scholar
  18. M. Ma, F. Zhuang, X. Hu et al., “Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/biotin-donor DNA system,” Cell Research, vol. 27, no. 4, pp. 578–581, 2017. View at: Publisher Site | Google Scholar
  19. E. J. Aird, K. N. Lovendahl, A. S. Martin, R. S. Harris, and W. R. Gordon, “Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template,” Communications Biology, vol. 1, no. 1, p. 54, 2018. View at: Publisher Site | Google Scholar
  20. A. V. Anzalone, P. B. Randolph, J. R. Davis et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature, vol. 576, no. 7785, pp. 149–157, 2019. View at: Publisher Site | Google Scholar
  21. R. Xu, J. Li, X. Liu, T. Shan, R. Qin, and P. Wei, “Development of plant prime-editing systems for precise genome editing,” Plant Communications, vol. 1, no. 3, p. 100043, 2020. View at: Publisher Site | Google Scholar
  22. H. Butt, G. S. Rao, K. Sedeek, R. Aman, R. Kamel, and M. Mahfouz, “Engineering herbicide resistance via prime editing in rice,” Plant Biotechnology Journal, 2020. View at: Publisher Site | Google Scholar
  23. X. Tang, S. Sretenovic, Q. Ren et al., “Plant prime editors enable precise gene editing in rice cells,” Molecular Plant, vol. 13, no. 5, pp. 667–670, 2020. View at: Publisher Site | Google Scholar
  24. Q. Lin, Y. Zong, C. Xue et al., “Prime genome editing in rice and wheat,” Nature Biotechnology, vol. 38, no. 5, pp. 582–585, 2020. View at: Publisher Site | Google Scholar
  25. H. Li, J. Li, J. Chen, L. Yan, and L. Xia, “Precise modifications of both exogenous and endogenous genes in rice by prime editing,” Molecular Plant, vol. 13, no. 5, pp. 671–674, 2020. View at: Publisher Site | Google Scholar
  26. J. Li, H. Li, J. Chen, L. Yan, and L. Xia, “Toward precision genome editing in crop plants,” Molecular Plant, vol. 13, no. 6, pp. 811–813, 2020. View at: Publisher Site | Google Scholar
  27. J. Strecker, A. Ladha, Z. Gardner et al., “RNA-guided DNA insertion with CRISPR-associated transposases,” Science, vol. 365, no. 6448, pp. 48–53, 2019. View at: Publisher Site | Google Scholar

Copyright © 2020 Yingxiao Zhang and Yiping Qi. Exclusive Licensee Nanjing Agricultural University. Distributed under a Creative Commons Attribution License (CC BY 4.0).

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