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SCIENCE CHINA Life Sciences, Volume 61, Issue 11: 1293-1300(2018) https://doi.org/10.1007/s11427-018-9392-7

Manipulating mRNA splicing by base editing in plants

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  • ReceivedSep 14, 2018
  • AcceptedSep 20, 2018
  • PublishedSep 27, 2018

Abstract

Precursor-mRNAs (pre-mRNA) can be processed into one or more mature mRNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intron-mediated enhancement (IME), whereas alternative splicing (AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB1.1 (encoding a type 2C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly, altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.


Funded by

grants from the National Key Research and Development Program of China(2016YFD0101804)

the National Natural Science Foundation of China(31788103,31420103912)

as well as the Chinese Academy of Sciences(QYZDY-SSW-SMC030,GJHZ1602)


Acknowledgment

This work was supported by grants from the National Key Research and Development Program of China (2016YFD0101804), the National Natural Science Foundation of China (31788103 and 31420103912), as well as the Chinese Academy of Sciences (QYZDY-SSW-SMC030 and GJHZ1602).


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1 Alignment of the cDNA sequences of AtHAB1(AT1G72770) wild type (WT) and mutant.

Figure S2 Alignment of the cDNA sequences of AtT30G6.16 (AT5G36290) wild type (WT) and mutant.

Figure S3 Comparison of AtT30G6.16 transcript levels in wild type (WT) and mutant.

Figure S4 Alignment of the cDNA sequences of AtRS31A (AT2G46610) wild type (WT) and mutant.

Figure S5 Online expression data for AtRS31A in the Arabidopsis eFP browser.

Figure S6 Alignment of the cDNA sequences of AtACT2 (AT3G18780) wild type (WT) and mutant.

Table S1 Molecular and genetic analysis of CRISPR/Cas9-induced splicing site mutations of AtHAB1, AtT30G6.16, AtRS31A and AtAct2 in T0 generation of Abadidopsis and their transmission to T1 generation

Table S2 Potential off-target sites analyzed for AtHAB1, AtT30G6.16, AtRS31A and AtAct2 in Arabidopsis

Table S3 Target sites of the sgRNAs in the 5′ splice site of the four studied genes, and the oligonucleotides to construct each sgRNA

Table S4 List of the PCR primers used in this study

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Base editing affects HAB1 splicing. A, Schematic representations of the influence of base editing on HAB1 splicing. Left part, the base editor target sequence in HAB1 and schematic diagram outlining HAB1 splicing in WT. Right part, the base-edited sequence in HAB1 and schematic diagram outlining HAB1 splicing in mutant. B, RT-PCR analysis of HAB1 mRNA variants in WT and mutant. “M” stands for DNA molecular weight ladder. C, Schematic diagram of HAB1 mRNA variants in WT and mutant and details of the sequence of RT-PCR amplicons from B. D, E and F, Sensitivity of WT and mutant seed to ABA treatment. Seeds were grown on half-strength MS medium supplemented with or without 0.25 µmol L–1 ABA. D, Images were captured 5 d after stratification. Scale bar, 0.2 cm. E and F, Cotyledon greening rates and percentage of plants with true leaves were separately recorded 4 and 7 d after stratification. (n=3 biologically independent experiments). All values represent means±SD. ****, P<0.0001; ns, no significant difference by two-tailed Student’s test.

  • Figure 2

    Base editing can alter T30G6.16 splicing. A, Schematic representations of the influence of base editing on T30G6.16 splicing. Left, the target sequence in T30G6.16, and a schematic diagram of T30G6.16 splicing in WT. Right, the base-edited sequence in t30g6.16.1 and a diagram of t30g6.16.1 splicing in the mutant. B, RT-PCR analysis of T30G6.16 mRNAs in WT and mutant. “M” is a DNA molecular weight ladder. C, Diagram of T30G6.16 mRNAs in WT and mutant, and details of the sequence of the RT-PCR amplicons from B.

  • Figure 3

    Base editing affects RS31A splicing. A, Schematic representations of the influence of base editing on RS31A splicing. Left part, the base editor target sequence in RS31A and schematic diagram outlining RS31A splicing in WT. Right part, the base-edited sequence in RS31A and schematic diagram outlining RS31A splicing in mutant. B, RT-PCR analysis of RS31A mRNA variants in WT and mutant. “M” stands for DNA molecular weight ladder. C, Schematic diagram of RS31A mRNA variants in WT and mutant and details of the sequence of RT-PCR amplicons from B. D, E and F, Sensitivity of WT and mutant seed to mitomycin C treatment. Seeds were grown on half-strength MS medium supplemented with or without 40 µmol L–1 mitomycin C. D, Images were captured 3 d after stratification. Scale bar, 0.2 cm. E and F, Cotyledon greening rates and percentage of seedling with true leave were separately recorded 3 and 8 d after stratification. (n=3 biologically independent experiments). All values represent means±SD. **, P< 0.01; ***, P<0.001; ns, no significant difference by two-tailed Student’s test.

  • Figure 4

    Base editing affects Act2 splicing. A, Schematic representations of the influence of base editing on Act2 splicing. Left part, the base editor target sequence in Act2 and schematic diagram outlining Act2 splicing in WT. Right part, the base-edited sequence in Act2 and schematic diagram outlining Act2 splicing in mutant. B, RT-PCR analysis of Act2 mRNA variants in WT and mutant. “M” stands for DNA molecular weight ladder. C, Schematic diagram of Act2 mRNA variants in WT and mutant and details of the sequence of RT-PCR amplicons from B. D, qPCR analysis of total Act2 transcript level in WT and mutant. All values represent means±SD. *, P< 0.05 (n=3 biologically independent experiments).

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