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Targeted Genome Editing in Genes and cis-Regulatory Regions Improves Qualitative and Quantitative Traits in Crops
Accepted Manuscript Targeted Genome Editing in Genes and Cis-Regulatory Regions Improves Qualitative and Quantitative Traits in Crops Xitao Li, Yongyao Xie, Qinlong Zhu, Yao-Guang Liu
PII: DOI: Reference:
S1674-2052(17)30308-8 10.1016/j.molp.2017.10.009 MOLP 537
To appear in: MOLECULAR PLANT Accepted Date: 20 October 2017
Please cite this article as: Li X., Xie Y., Zhu Q., and Liu Y.-G. (2017). Targeted Genome Editing in Genes and Cis-Regulatory Regions Improves Qualitative and Quantitative Traits in Crops. Mol. Plant. doi: 10.1016/j.molp.2017.10.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
ACCEPTED MANUSCRIPT Title: Targeted Genome Editing in Genes and Cis-Regulatory Regions Improves Qualitative and Quantitative Traits in Crops
The Clustered regularly interspaced short palindromic repeats (CRISPR)-associated
RI PT
protein 9 (CRISPR/Cas9)-based genome editing system is a revolutionary technology for targeted mutagenesis in molecular biology research and genetic improvement of traits in crops (Cong et al., 2013; Ma et al., 2015; Ma et al., 2016). Agronomic traits of crops are controlled by major genes and quantitative trait loci (QTL). Therefore,
SC
the CRISPR/Cas9 system can be used to effectively and rapidly produce mutant traits by different strategies (Figure 1A-C). The most common application of the targeted
M AN U
editing system in genetic improvement is to knock out completely the functions of target genes, usually by editing site(s) in the coding sequences (CDS) to produce null-allele mutants (Figure 1A). For instance, CRISPR/Cas9-based knocking-out of SaF+ or SaM+ and OgTPR1 at the hybrid sterility loci Sa and S1, respectively, could produce neutral alleles and hybrid-compatible rice lines for overcoming hybrid
TE D
sterility in inter-subspecific and inter-specific hybrid rice breeding (Xie et al., 2017a; Xie et al., 2017b). Other cases include the knocking-out of TMS5 in rice could rapidly breed temperature-sensitive male-sterile lines used for “two-line” hybrid rice breeding
EP
(Zhou et al., 2016), and knocking-out of GS3 and Gn1a generated different effects on variations of grain size, grain number, and tiller number in different genetic
AC C
backgrounds (Shen et al., 2016). Besides, the genome editing technology also enables knocking out a copy or copies of duplicated genes to create artificial copy number variation, which produces gene dosage effect on phenotypic variation (Figure 1B). This application has been recently demonstrated on studying the hybrid male sterility locus Sc in rice, in which reduction of the tandem-repeated gene copy number in indica rice allele Sc-i by CRISPR/Cas9 could improve male fertility in japonica-indica hybrids (Shen et al., 2017). However, current genetic improvement of agronomic traits mainly relies on the time-consuming and laborious selection and introgression of rare natural mutations in 1
ACCEPTED MANUSCRIPT QTLs, many of which occurred in the regulatory regions of promoters (Wittkopp and Kalay, 2011). The cis-regulatory elements (CREs) in promoter regions, which are integral part of the genes, regulate gene expression at the right time and level, and proper spatial distribution. Unlike mutations in coding sequences that may destroy
RI PT
completely the gene functions and result in null-allele (loss-of-function) mutants, mutations in cis-regulatory regions often cause change of the expression level and/or pattern of the genes, thus leading to quantitative variation of the traits, (Wittkopp and Kalay, 2011). Therefore, it has great potential to exploit high-effective approaches
SC
using the CRISPR/Cas9(Cpf1) genome editing systems for producing large number of cis-regulatory mutant alleles of target genes (Figure 1C). Now, Rodríguez-Leal et al.
M AN U
(2017) have used the CRISPR/Cas9 system to modify the promoters of target genes that control fruit size, inflorescence architecture, and plant growth habit, to generate numerous consecutive variations of these traits in tomato (Solanum lycopersicum). CLAVATA-WUSCHEL stem cell circuit (CLV-WUS) mediates a classical signaling pathway for regulating meristem size in plants (Somssich et al., 2016).
TE D
Rodríguez-Leal et al. (2017) found that CRISPR/Cas9-based mutations (small fragmental deletions) in the downstream and promoter upstream regions (containing known and unknown CREs) of the tomato WUS (SlWUS) and CLV3 (SlCLV3) genes,
EP
respectively, could generate novel cis-regulatory mutant alleles, which increased fruit locule number and fruit size, similar to the natural QTL variants, indicating that it is feasible to generate engineered QTLs by genome editing. Then, by crossing a
AC C
transgenic plant carrying the CRISPR/Cas9 multiplex-editing components (the Cas9 and sgRNA cassettes for targeting eight sites in the promoter region of SlCLV3) with a wild-type line to obtain hundreds of mutant plants, they developed a CRISPR/Cas9-driven genetic screen approach to rapidly generate and evaluate large number of SlCLV3-promoter mutant alleles for quantitative variation of fruit locule number. To further expand the utilization of the CRISPR/Cas9-based cis-regulatory mutagenesis approach, the authors also edited the promoter regions of the genes, COMPOUND INFLORESCENCE (S) that controls the inflorescence architecture, and SELF PRUNING (SP) that functions in plant architecture. As expected, they obtained 2
ACCEPTED MANUSCRIPT various novel S- and SP-promoter mutant alleles, which resulted in quantitative phenotypic variation of the traits (Rodríguez-Leal et al., 2017). Although this mutagenesis approach has no methodological innovation in the genome editing system, it provides good examples for creating artificial variation of
RI PT
quantitative traits by targeted editing of CRE-containing sequences, thus promoting molecular breeding in crops. The advantages and significance of this approach embody in following aspects. First, novel QTLs resulted from altered expression levels and patterns can be effectively produced by using the CRISPR/Cas9-based
SC
cis-regulatory mutagenesis. Natural QTL variants from spontaneous mutations have evolved by over thousand even million years, but they have resulted in limited range
M AN U
of changes in agronomic traits. The plant breeder's goal is to find and use natural QTL variants to improve crop productivity, but it is very time-consuming and laborious. By editing of CREs in the promoter (and other) regions of target genes, as demonstrated by Rodríguez-Leal et al. (2017), it is highly effective to create numerous allele variants that may produce better phenotypes. In addition, the transgenic components
TE D
(Cas9 and the antibiotic-selectable marker gene) can be simply removed by segregation in the progenies, producing transgene-free gene-edited plant lines. Second, the phenotypic effect of the cis-regulatory mutant alleles is often unpredictable; this
EP
phenotypic flexibility of the variants can provide more abundant diversity for genetic improvement in crops. Third, this mutagenesis approach is easy to follow for molecular breeding in other crops, such as rice, corn, sorghum and wheat. Finally, a
AC C
large amount of regulatory mutations created by this approach can systematically reveal the association of CREs with phenotypic variation, thus facilitating dissecting their functions and spatial organization. At present the genome editing technology is mainly used for targeted mutations
of specific loci (Figure 1). This is useful for improving some agronomic traits by knocking out and partially enhancing or weakening the functionality of negative regulatory genes. However, the current gene editing technology is still inefficient in plants for modifying genes by precise editing, i.e., fixed-point base substitution and fragment insertion/deletion/replacement, although a few successful cases of precise 3
ACCEPTED MANUSCRIPT editing in plants have been reported, for example, the targeted insertion or replacement of the maize GOS2 promoter in the upstream regions of the open reading frame of the maize ARGOS8 gene could elevate the gene expression, thus improving yield under drought stress (Shi et al., 2017). Therefore, it is a big challenge to exploit
RI PT
effective precise editing technologies for basic research as well as genetic improvement of many important agronomic traits of crops in the future.
In summary, the CRISPR/Cas9-based multiplex cis-regulatory mutagenesis approach expands the application range of the genome editing technology to elaborate
SC
quantitative traits in crop breeding.
M AN U
FUNDING
This research was supported by grants from Guangdong Province Public Interest Research and Capacity Building Special Fund (2015B020201002), and the Ministry of Agriculture of the People’s Republic of China (2016ZX08010-001,
TE D
2016ZX08009-002).
ACKNOWLEDGMENTS
We apologize for not citing all the relevant references due to space limitations. No conflict of interest declared.
1
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Xitao Li1, 2,3, Yongyao Xie1, 3, Qinlong Zhu1, 3, Yao-Guang Liu1, 2,3,* State Key Laboratory for Conservation and Utilization of Subtropical
2
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Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China. Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong
Provincial Higher Education Institutions, South China Agricultural University, Guangzhou 510642, China. 3
College of Life Sciences, South China Agricultural University, Guangzhou 510642,
China * Corresponding authors ([email protected])
REFERENCES 4
ACCEPTED MANUSCRIPT Cong. L., Ran. F. A., Cox. D., Lin. S., Barretto. R., Habib. N., Hsu. P. D., Wu. X., Jiang. W., Marraffini. L. A., Zhang. F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 15: 819-823. Ma X, Zhu Q, Chen Y, Liu Y-G. (2016). CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol. Plant 9: 961–974.
RI PT
Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., Wang, B., Yang, Z., Li, H., Lin, Y. et al. (2015). A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant 8: 1274-1284. Rodríguez-Leal, D.， ， Lemmon， ， Z. H., Man, J.， ， Bartlett， ，M. E., Lippman, Z. B. (2017). Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing. Cell DOI:10.1016/j.cell.2017.08.030.
M AN U
SC
Wittkopp, P.J., and Kalay, G. (2011). Cis-regulatory elements: Molecular mechanisms and evolutionary processes underlying divergence. Nat. Rev. Genet. 13: 59-69. Shi, J., Gao, H., Wang, H., Lafitte, H.R., Archibald, R.L., Yang, M., Hakimi, S.M., Mo, H., and Habben, J.E. (2017). ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotech. J. 15:207-216.
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Shen, L., Wang, C., Fu, Y., Wang, J., Liu, Q., Zhang, X., Yan, C., Qian, Q., and Wang, K. (2016). QTL editing confers opposing yield performance in different rice varieties. J. Integr. Plant Biol. doi: 10.1111/jipb.12501 Shen, R., Wang, L., Liu, X., Wu, J., Jin, W., Zhao, X., Xie, X., Zhu, Q., Tang, H., Li, Q., et al. (2017). Genomic structural variation-mediated allelic suppression causes hybrid male sterility in rice. Nature Com. DOI: 10.1038/s41467-017-01400-y.
EP
Somssich, M., Je, B.I., Simon, R., and Jackson, D. (2016). CLAVATAWUSCHEL signaling in the shoot meristem. Development 143: 3238-3248.
AC C
Xie, Y., Niu, B., Long, Y., Li, G., Tang, J., Zhang, Y., Xie, X., Ren, D., Liu, Y.-G., Chen, L. (2017a). Suppression or knockout of SaF/SaM overcomes the Sa-mediated hybrid male sterility in rice. J. Integr. Plant Biol. 9: 669-679. Xie, Y., Xu P., Huang, J., Ma, S., Xie, X., Tao, D., Chen, L., Liu, Y.-G. (2017b). Interspecific hybrid sterility in rice is mediated by OgTPR1 at the S1 locus encoding a peptidase-like protein. Mol. Plant. 10:1137-1140. Zhou, H.， ， He, M.， ， Li, J.， ， Chen, L.， ， Huang, Z.， ， Zheng, S.， ，Zhu, L.， ，Ni, E.， ，Jiang, D.， ，Zhao, B.， ，et al. (2016). Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system. Sci Rep. 6: 37395
5
ACCEPTED MANUSCRIPT Figure legend
Figure 1 Strategies for removing and modifying gene function by the genome editing technology. (A) Targeted editing at a site or sites (small arrows) in the gene coding sequence
RI PT
(CDS) using a genome editing system usually produces frame-shift, premature stop codon, or fragment deletion, thus destroys completely the gene function and causes phenotypic variation of the trait.
(B) For duplicated gene copies (A1, A2 or more copies) that have gene dosage effect
SC
on the trait, selective knocking-out of part of the copies by targeted editing (in CDS, or deletion of the whole gene sequence as indicated by a dot-line) can
M AN U
generate gene dosage-reduced mutant alleles that may cause phenotypic variation. Asterisk indicates varied nucleotide(s) that can be used to design a specific target-site/PAM (protospacer adjacent motif) for editing in A2 but not in A1. (C) Targeted editing at one, two, or multiple sites in the promoter region containing cis-regulatory elements (CREs) to generate multiple cis-regulatory mutant alleles,
TE D
which may alter the expression level and/or pattern of the gene and produce quantitative variation of the trait. Destroyed CREs and deleted sequences are
AC C
EP
shown by grayed marks and dot-lines, respectively.
6
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EP
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M AN U
SC
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ACCEPTED MANUSCRIPT
PII: DOI: Reference:
S1674-2052(17)30308-8 10.1016/j.molp.2017.10.009 MOLP 537
To appear in: MOLECULAR PLANT Accepted Date: 20 October 2017
Please cite this article as: Li X., Xie Y., Zhu Q., and Liu Y.-G. (2017). Targeted Genome Editing in Genes and Cis-Regulatory Regions Improves Qualitative and Quantitative Traits in Crops. Mol. Plant. doi: 10.1016/j.molp.2017.10.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
ACCEPTED MANUSCRIPT Title: Targeted Genome Editing in Genes and Cis-Regulatory Regions Improves Qualitative and Quantitative Traits in Crops
The Clustered regularly interspaced short palindromic repeats (CRISPR)-associated
RI PT
protein 9 (CRISPR/Cas9)-based genome editing system is a revolutionary technology for targeted mutagenesis in molecular biology research and genetic improvement of traits in crops (Cong et al., 2013; Ma et al., 2015; Ma et al., 2016). Agronomic traits of crops are controlled by major genes and quantitative trait loci (QTL). Therefore,
SC
the CRISPR/Cas9 system can be used to effectively and rapidly produce mutant traits by different strategies (Figure 1A-C). The most common application of the targeted
M AN U
editing system in genetic improvement is to knock out completely the functions of target genes, usually by editing site(s) in the coding sequences (CDS) to produce null-allele mutants (Figure 1A). For instance, CRISPR/Cas9-based knocking-out of SaF+ or SaM+ and OgTPR1 at the hybrid sterility loci Sa and S1, respectively, could produce neutral alleles and hybrid-compatible rice lines for overcoming hybrid
TE D
sterility in inter-subspecific and inter-specific hybrid rice breeding (Xie et al., 2017a; Xie et al., 2017b). Other cases include the knocking-out of TMS5 in rice could rapidly breed temperature-sensitive male-sterile lines used for “two-line” hybrid rice breeding
EP
(Zhou et al., 2016), and knocking-out of GS3 and Gn1a generated different effects on variations of grain size, grain number, and tiller number in different genetic
AC C
backgrounds (Shen et al., 2016). Besides, the genome editing technology also enables knocking out a copy or copies of duplicated genes to create artificial copy number variation, which produces gene dosage effect on phenotypic variation (Figure 1B). This application has been recently demonstrated on studying the hybrid male sterility locus Sc in rice, in which reduction of the tandem-repeated gene copy number in indica rice allele Sc-i by CRISPR/Cas9 could improve male fertility in japonica-indica hybrids (Shen et al., 2017). However, current genetic improvement of agronomic traits mainly relies on the time-consuming and laborious selection and introgression of rare natural mutations in 1
ACCEPTED MANUSCRIPT QTLs, many of which occurred in the regulatory regions of promoters (Wittkopp and Kalay, 2011). The cis-regulatory elements (CREs) in promoter regions, which are integral part of the genes, regulate gene expression at the right time and level, and proper spatial distribution. Unlike mutations in coding sequences that may destroy
RI PT
completely the gene functions and result in null-allele (loss-of-function) mutants, mutations in cis-regulatory regions often cause change of the expression level and/or pattern of the genes, thus leading to quantitative variation of the traits, (Wittkopp and Kalay, 2011). Therefore, it has great potential to exploit high-effective approaches
SC
using the CRISPR/Cas9(Cpf1) genome editing systems for producing large number of cis-regulatory mutant alleles of target genes (Figure 1C). Now, Rodríguez-Leal et al.
M AN U
(2017) have used the CRISPR/Cas9 system to modify the promoters of target genes that control fruit size, inflorescence architecture, and plant growth habit, to generate numerous consecutive variations of these traits in tomato (Solanum lycopersicum). CLAVATA-WUSCHEL stem cell circuit (CLV-WUS) mediates a classical signaling pathway for regulating meristem size in plants (Somssich et al., 2016).
TE D
Rodríguez-Leal et al. (2017) found that CRISPR/Cas9-based mutations (small fragmental deletions) in the downstream and promoter upstream regions (containing known and unknown CREs) of the tomato WUS (SlWUS) and CLV3 (SlCLV3) genes,
EP
respectively, could generate novel cis-regulatory mutant alleles, which increased fruit locule number and fruit size, similar to the natural QTL variants, indicating that it is feasible to generate engineered QTLs by genome editing. Then, by crossing a
AC C
transgenic plant carrying the CRISPR/Cas9 multiplex-editing components (the Cas9 and sgRNA cassettes for targeting eight sites in the promoter region of SlCLV3) with a wild-type line to obtain hundreds of mutant plants, they developed a CRISPR/Cas9-driven genetic screen approach to rapidly generate and evaluate large number of SlCLV3-promoter mutant alleles for quantitative variation of fruit locule number. To further expand the utilization of the CRISPR/Cas9-based cis-regulatory mutagenesis approach, the authors also edited the promoter regions of the genes, COMPOUND INFLORESCENCE (S) that controls the inflorescence architecture, and SELF PRUNING (SP) that functions in plant architecture. As expected, they obtained 2
ACCEPTED MANUSCRIPT various novel S- and SP-promoter mutant alleles, which resulted in quantitative phenotypic variation of the traits (Rodríguez-Leal et al., 2017). Although this mutagenesis approach has no methodological innovation in the genome editing system, it provides good examples for creating artificial variation of
RI PT
quantitative traits by targeted editing of CRE-containing sequences, thus promoting molecular breeding in crops. The advantages and significance of this approach embody in following aspects. First, novel QTLs resulted from altered expression levels and patterns can be effectively produced by using the CRISPR/Cas9-based
SC
cis-regulatory mutagenesis. Natural QTL variants from spontaneous mutations have evolved by over thousand even million years, but they have resulted in limited range
M AN U
of changes in agronomic traits. The plant breeder's goal is to find and use natural QTL variants to improve crop productivity, but it is very time-consuming and laborious. By editing of CREs in the promoter (and other) regions of target genes, as demonstrated by Rodríguez-Leal et al. (2017), it is highly effective to create numerous allele variants that may produce better phenotypes. In addition, the transgenic components
TE D
(Cas9 and the antibiotic-selectable marker gene) can be simply removed by segregation in the progenies, producing transgene-free gene-edited plant lines. Second, the phenotypic effect of the cis-regulatory mutant alleles is often unpredictable; this
EP
phenotypic flexibility of the variants can provide more abundant diversity for genetic improvement in crops. Third, this mutagenesis approach is easy to follow for molecular breeding in other crops, such as rice, corn, sorghum and wheat. Finally, a
AC C
large amount of regulatory mutations created by this approach can systematically reveal the association of CREs with phenotypic variation, thus facilitating dissecting their functions and spatial organization. At present the genome editing technology is mainly used for targeted mutations
of specific loci (Figure 1). This is useful for improving some agronomic traits by knocking out and partially enhancing or weakening the functionality of negative regulatory genes. However, the current gene editing technology is still inefficient in plants for modifying genes by precise editing, i.e., fixed-point base substitution and fragment insertion/deletion/replacement, although a few successful cases of precise 3
ACCEPTED MANUSCRIPT editing in plants have been reported, for example, the targeted insertion or replacement of the maize GOS2 promoter in the upstream regions of the open reading frame of the maize ARGOS8 gene could elevate the gene expression, thus improving yield under drought stress (Shi et al., 2017). Therefore, it is a big challenge to exploit
RI PT
effective precise editing technologies for basic research as well as genetic improvement of many important agronomic traits of crops in the future.
In summary, the CRISPR/Cas9-based multiplex cis-regulatory mutagenesis approach expands the application range of the genome editing technology to elaborate
SC
quantitative traits in crop breeding.
M AN U
FUNDING
This research was supported by grants from Guangdong Province Public Interest Research and Capacity Building Special Fund (2015B020201002), and the Ministry of Agriculture of the People’s Republic of China (2016ZX08010-001,
TE D
2016ZX08009-002).
ACKNOWLEDGMENTS
We apologize for not citing all the relevant references due to space limitations. No conflict of interest declared.
1
EP
Xitao Li1, 2,3, Yongyao Xie1, 3, Qinlong Zhu1, 3, Yao-Guang Liu1, 2,3,* State Key Laboratory for Conservation and Utilization of Subtropical
2
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Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China. Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong
Provincial Higher Education Institutions, South China Agricultural University, Guangzhou 510642, China. 3
College of Life Sciences, South China Agricultural University, Guangzhou 510642,
China * Corresponding authors ([email protected])
REFERENCES 4
ACCEPTED MANUSCRIPT Cong. L., Ran. F. A., Cox. D., Lin. S., Barretto. R., Habib. N., Hsu. P. D., Wu. X., Jiang. W., Marraffini. L. A., Zhang. F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 15: 819-823. Ma X, Zhu Q, Chen Y, Liu Y-G. (2016). CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol. Plant 9: 961–974.
RI PT
Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., Wang, B., Yang, Z., Li, H., Lin, Y. et al. (2015). A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant 8: 1274-1284. Rodríguez-Leal, D.， ， Lemmon， ， Z. H., Man, J.， ， Bartlett， ，M. E., Lippman, Z. B. (2017). Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing. Cell DOI:10.1016/j.cell.2017.08.030.
M AN U
SC
Wittkopp, P.J., and Kalay, G. (2011). Cis-regulatory elements: Molecular mechanisms and evolutionary processes underlying divergence. Nat. Rev. Genet. 13: 59-69. Shi, J., Gao, H., Wang, H., Lafitte, H.R., Archibald, R.L., Yang, M., Hakimi, S.M., Mo, H., and Habben, J.E. (2017). ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotech. J. 15:207-216.
TE D
Shen, L., Wang, C., Fu, Y., Wang, J., Liu, Q., Zhang, X., Yan, C., Qian, Q., and Wang, K. (2016). QTL editing confers opposing yield performance in different rice varieties. J. Integr. Plant Biol. doi: 10.1111/jipb.12501 Shen, R., Wang, L., Liu, X., Wu, J., Jin, W., Zhao, X., Xie, X., Zhu, Q., Tang, H., Li, Q., et al. (2017). Genomic structural variation-mediated allelic suppression causes hybrid male sterility in rice. Nature Com. DOI: 10.1038/s41467-017-01400-y.
EP
Somssich, M., Je, B.I., Simon, R., and Jackson, D. (2016). CLAVATAWUSCHEL signaling in the shoot meristem. Development 143: 3238-3248.
AC C
Xie, Y., Niu, B., Long, Y., Li, G., Tang, J., Zhang, Y., Xie, X., Ren, D., Liu, Y.-G., Chen, L. (2017a). Suppression or knockout of SaF/SaM overcomes the Sa-mediated hybrid male sterility in rice. J. Integr. Plant Biol. 9: 669-679. Xie, Y., Xu P., Huang, J., Ma, S., Xie, X., Tao, D., Chen, L., Liu, Y.-G. (2017b). Interspecific hybrid sterility in rice is mediated by OgTPR1 at the S1 locus encoding a peptidase-like protein. Mol. Plant. 10:1137-1140. Zhou, H.， ， He, M.， ， Li, J.， ， Chen, L.， ， Huang, Z.， ， Zheng, S.， ，Zhu, L.， ，Ni, E.， ，Jiang, D.， ，Zhao, B.， ，et al. (2016). Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system. Sci Rep. 6: 37395
5
ACCEPTED MANUSCRIPT Figure legend
Figure 1 Strategies for removing and modifying gene function by the genome editing technology. (A) Targeted editing at a site or sites (small arrows) in the gene coding sequence
RI PT
(CDS) using a genome editing system usually produces frame-shift, premature stop codon, or fragment deletion, thus destroys completely the gene function and causes phenotypic variation of the trait.
(B) For duplicated gene copies (A1, A2 or more copies) that have gene dosage effect
SC
on the trait, selective knocking-out of part of the copies by targeted editing (in CDS, or deletion of the whole gene sequence as indicated by a dot-line) can
M AN U
generate gene dosage-reduced mutant alleles that may cause phenotypic variation. Asterisk indicates varied nucleotide(s) that can be used to design a specific target-site/PAM (protospacer adjacent motif) for editing in A2 but not in A1. (C) Targeted editing at one, two, or multiple sites in the promoter region containing cis-regulatory elements (CREs) to generate multiple cis-regulatory mutant alleles,
TE D
which may alter the expression level and/or pattern of the gene and produce quantitative variation of the trait. Destroyed CREs and deleted sequences are
AC C
EP
shown by grayed marks and dot-lines, respectively.
6
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EP
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M AN U
SC
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ACCEPTED MANUSCRIPT