SpCas9-NG self-targets the sgRNA sequence in plant genome editing

Abstract

Streptococcus pyogenes Cas9 (SpCas9)-NG recognizes NGN protospacer adjacent motifs and expands the scope of genome-editing tools. In this study, we found that SpCas9-NG not only targeted the genome but also efficiently self-targeted the single-guide RNA sequence in transfer DNA in transgenic plants, potentially increasing off-target risk by generating new single-guide RNAs. We further showed that the self-target effect of SpCas9-NG could be greatly alleviated by using a modified single-guide RNA scaffold starting with a GCCCC sequence.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: CRISPR-SpCas9-NG targeting the genome and sgRNA in transgenic rice.
Fig. 2: Self-targeting of SpCas9-NG induced secondary editing and could be alleviated using sgRNA scaffold starting with GCCCC.

Data availability

The data sets generated in the study are available from the corresponding authors on reasonable request. Source data for Fig. 1 and Extended Data Figs. 24 are provided with the paper.

References

  1. 1.

    Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    CAS  Article  Google Scholar 

  2. 2.

    Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    CAS  Article  Google Scholar 

  3. 3.

    Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).

    CAS  Article  Google Scholar 

  4. 4.

    Endo, M. et al. Genome editing in plants by engineered CRISPR–Cas9 recognizing NG PAM. Nat. Plants 5, 14–17 (2019).

    CAS  Article  Google Scholar 

  5. 5.

    Ge, Z. et al. Engineered xCas9 and SpCas9-NG variants broaden PAM recognition sites to generate mutations in Arabidopsis plants. Plant Biotechnol. J. 17, 1865–1867 (2019).

    Article  Google Scholar 

  6. 6.

    Hua, K., Tao, X., Han, P., Wang, R. & Zhu, J.-K. Genome engineering in rice using Cas9 variants that recognize NG PAM sequences. Mol. Plant 12, 1003–1014 (2019).

    CAS  Article  Google Scholar 

  7. 7.

    Ren, B. et al. Cas9-NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice. Mol. Plant 12, 1015–1026 (2019).

    CAS  Article  Google Scholar 

  8. 8.

    Zhong, Z. et al. Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG. Mol. Plant 12, 1027–1036 (2019).

    CAS  Article  Google Scholar 

  9. 9.

    Xing, H.-L. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 14, 327 (2014).

    Article  Google Scholar 

  10. 10.

    Kelliher, T. et al. One-step genome editing of elite crop germplasm during haploid induction. Nat. Biotechnol. 37, 287–292 (2019).

    CAS  Article  Google Scholar 

  11. 11.

    Filichkin, S. A. & Gelvin, S. B. Formation of a putative relaxation intermediate during T-DNA processing directed by the Agrobacterium tumefaciens VirD1, D2 endonuclease. Mol. Microbiol. 8, 915–926 (1993).

    CAS  Article  Google Scholar 

  12. 12.

    Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

    CAS  Article  Google Scholar 

  13. 13.

    Xu, R.-F. et al. Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Sci. Rep. 5, 11491 (2015).

    CAS  Article  Google Scholar 

  14. 14.

    Wang, Z.-P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144 (2015).

    Article  Google Scholar 

  15. 15.

    Dang, Y. et al. Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency. Genome Biol. 16, 280 (2015).

    Article  Google Scholar 

  16. 16.

    Qin, R. et al. Developing a highly efficient and wildly adaptive CRISPR-SaCas9 toolset for plant genome editing. Plant Biotechnol. J. 17, 706–708 (2019).

    Article  Google Scholar 

  17. 17.

    Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441–443 (2017).

    CAS  Article  Google Scholar 

  18. 18.

    Li, H. et al. CRISPR/Cas9-mediated adenine base editing in rice genome. Rice Sci. 26, 125–128 (2019).

    Article  Google Scholar 

  19. 19.

    Hu, L. et al. Plant phosphomannose isomerase as a selectable marker for rice transformation. Sci. Rep. 6, 25921 (2016).

    CAS  Article  Google Scholar 

  20. 20.

    Liu, Q. et al. Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems. Sci. China Life Sci. 62, 1–7 (2019).

    Article  Google Scholar 

Download references

Acknowledgements

This work was funded by the Genetically Modified Breeding Major Projects (grant nos. 2019ZX08010003-001-008 and 2016ZX08010-002-008) and the National Natural Science Foundation of China (grant no. U19A2022).

Author information

Affiliations

Authors

Contributions

P.W. conceived this study. P.W. and J.Y. supervised the research. R.Q. and J.L. performed all experiments with help from the other authors. X.L. contributed to rice transformations. R.X. contributed to plant genotyping, protein expression and in vitro cleavage assay. P.W. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Jianbo Yang or Pengcheng Wei.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Seiichi Toki, Bing Yang and the other, anonymous, reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Targets of presumed self-edited sgRNAs could be mutated by SpCas9-NG in rice.

As described in Fig. 2a, the potential targets of presumed sgRNA mutants that might be generated from the self-editing of the sgRNA by SpCas9-NG were tandemly arranged in a synthesized sequence and inserted into the SpCas9-NG vector. The mutations at the inserted targets were genotyped in transgenic plants of PDS-1 sgRNA (a) or LAZY-1 sgRNA (b) and aligned. Blue, protospacer sequence; red, deletions or insertions. The PAM is underlined. The genotype of the mutation is indicated on the right of the sequence.

Extended Data Fig. 2 The mutated sgRNA induced by SpCas9-NG-BE could mediate secondary editing in rice.

a, Schematic representation of vectors for validating the activity of the self-edited sgRNA by SpCas9-NG-BE. A rice genome sequence containing a synthesized target of presumed self-edited sgRNA (mTarget) was inserted into the SpCas9-NG-eBE3 vector. The same fragment was also inserted into SpCas9-eBE3 as a control. Arrows indicate the primers used to detect possible self-editing of sgRNA and the secondary editing by the presumed sgRNA mutant. b, Sequences of the guide RNA and inserted mTarget in the T-DNA region. The guide sequence and PAM are labeled with blue and orange, respectively. The self-editing of sgRNA may lead to single-base conversion at the red letter for recognition of the mTarget. The restriction enzyme (RE) site is underlined. c, PCR-RE assay to detect self-editing of SpCas9-NG-eBE3 and the secondary editing by the mutated sgRNA in rice. The vectors were introduced into rice by Agrobacterium-mediated transformation. After 4 weeks of selection, more than 200 resistant events were collected together to isolate genomic DNA for the PCR-RE assay on the sgRNA and mTarget region. Left, the digestion band of SpCas9-NG-eBE3 implied that the sgRNA was self-edited, as expected. Right, the undigested bands of SpCas-NG-eBE3 suggested the occurrence of secondary editing by the mutated sgRNA and were extracted for clone sequencing. d, Sanger sequencing of the undigested mTarget band. The arrows indicate the secondary base editing that should be induced by the self-edited sgRNA. c, d, experiments were repeated at least three times and the similar results were obtained. Source data

Extended Data Fig. 3 In vitro cleavage assay of SpCas9-NG.

a, Expression and purification of SpCas9-NG in bacteria. The 6XHis tag was used for purification. SpCas9 was expressed in parallel and purified as a control. Experiments were repeated two times and the similar results were generated. b, A fragment containing the PDS-1 target was cloned from the OsPDS gene as an in vitro cleavage target. To test different PAMs, the TGG PAM of the PDS-1 target was mutated to TGT or GTT. Blue, guide sequence; orange, PAM. c, Electrophoresis of cleavage products. The target fragment was incubated with SpCas9 or SpCas9-NG protein and the in vitro transcribed sgRNA complex (50 nM) for 30 min. The fragments were separated by 3% agarose gel electrophoresis. The digested bands (red arrows) indicated that only the target with a TGG PAM could be cut by SpCas9, while all 3 targets could be cleaved by SpCas9-NG. d, Time-course assay of SpCas9-NG activity on different PAMs. The PDS-1 protospacer with a TGG, TGT or GTT PAM was used as target of SpCas9-NG in vitro cleavage. The reaction products were sampled at 0, 2, 5, 10, 15 and 30 min and analyzed by electrophoresis. c, d, experiments were repeated three times and the similar results were observed. Source data

Extended Data Fig. 4 Mutagenesis of T-DNA targets by SpCas9-NG.

a, Schematic representation of vectors used to examine mutation efficiency at the inserted targets in T-DNA. The genomic fragment containing the targets was cloned into the T-DNA of the pHUC411-NG vector with the respective sgRNA. b, Determining the mutagenesis efficiency of the same target sequences in plant genome or inserted T-DNA region. The vectors were separately transformed into rice by Agrobacterium-mediated transformation. After 4 weeks of selection, approximately 200 resistant calli were collected from one transformant as a sample. The targets in the genome, sgRNA and inserted sequence were examined by clone sequencing. For each target, 36 clones from one sample were tested, and the mutation efficiency was calculated as the ratio of mutated clones to total clones. Three biological replicates were performed. The mutagenesis efficiencies were compared between the target site in the genome and inserted site in T-DNA with exactly the same protospacer and PAM sequence by two tailed t-test analysis (**, p < 0.01). Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 1–9.

Reporting Summary

Source data

Source Data Fig. 1

Statistical Source Data.

Source Data Extended Data Fig. 2

Unprocessed gels.

Source Data Extended Data Fig. 3

Unprocessed gels.

Source Data Extended Data Fig. 4

Statistical Source Data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Qin, R., Li, J., Liu, X. et al. SpCas9-NG self-targets the sgRNA sequence in plant genome editing. Nat. Plants 6, 197–201 (2020). https://doi.org/10.1038/s41477-020-0603-9

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing