Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

doi:10.1016/j.rsci.2018.11.001

摘要/Abstract

关键词: genome editing, suicide gene, transgene killer CRISPR, Cas9, transgene-free

. [J]. Rice Science, 2019, 26(2): 109-117.

图/表 3

参考文献 56

[1]	Burstein D, Harrington L B, Strutt S C, Probst A J, Anantharaman K, Thomas B C, Doudna J A, Banfield J F.2017. New CRISPR- Cas systems from uncultivated microbes.Nature, 542: 237-241.
[2]	Casini A, Olivieri M, Petris G, Montagna C, Reginato G, Maule G, Lorenzin F, Prandi D, Romanel A, Demichelis F, Inga A, Cereseto A.2018. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat Biotechnol, 36(3): 265-271.
[3]	Cermak T, Baltes N J, Cegan R, Zhang Y, Voytas D F.2015. High-frequency, precise modification of the tomato genome.Genome Biol, 16: 232.
[4]	Chu V T, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R.2015. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.Nat Biotechnol, 33(5): 543-548.
[5]	Cong L, Ran F A, Cox D, Lin S L, Barretto R, Habib N, Hsu P D, Wu X B, Jiang W Y, Marraffini L A, Zhang F.2013. Multiplex genome engineering using CRISPR/Cas systems.Science, 339: 819-823.
[6]	Cook M, Thilmony R.2012. The OsGEX2 gene promoter confers sperm cell expression in transgenic rice. Plant Mol Biol Rep, 30(5): 1138-1148.
[7]	Dreissig S, Schiml S, Schindele P, Weiss O, Rutten T, Schubert V, Gladilin E, Mette M F, Puchta H, Houben A.2017. Live cell CRISPR-imaging in plants reveals dynamic telomere movements.Plant J, 91(4): 565-573.
[8]	Fonfara I, Richter H, Bratovic M, Le Rhun A, Charpentier E.2016. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA.Nature, 532: 517-521.
[9]	Gallego-Bartolome J, Gardiner J, Liu W L, Papikian A, Ghoshal B, Kuo H Y, Zhao J M C, Segal D J, Jacobsen S E.2018. Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain. Proc Natl Acad Sci USA, 115(9): 2125-2134.
[10]	Gao L, Cox D B T, Yan W X, Manteiga J C, Schneider M W, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F.2017. Engineered Cpf1 variants with altered PAM specificities.Nat Biotechnol, 35(8): 789-792.
[11]	Gao X H, Chen J L, Dai X H, Zhang D, Zhao Y D.2016. An effective strategy for reliably isolating heritable and cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Plant Physiol, 171(3): 1794-1800.
[12]	Gaudelli N M, Komor A C, Rees H A, Packer M S, Badran A H, Bryson D I, Liu D R.2017. Programmable base editing of AT to GC in genomic DNA without DNA cleavage.Nature, 551: 464-471.
[13]	Gibson D G, Young L, Chuang R Y, Venter J C, Hutchison C A, Smith H O.2009. Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Methods, 6(5): 343-345.
[14]	Gilbert L A, Larson M H, Morsut L, Liu Z, Brar G A, Torres S E, Stern-Ginossar N, Brandman O, Whitehead E H, Doudna J A, Lim W A, Weissman J S, Qi L S.2013. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.Cell, 154(2): 442-451.
[15]	Hall T C, Kumpatla S P, Kharb P, Iyer L, Cervera M, Jiang Y, Wang T, Yang G, Teerawanichpan P, Narangajavana J, Dong J.2001. Gene silencing and its reactivation in transgenic rice. In: Rice Genetics IV. World Scientific: 465-481.
[16]	He Y B, Wang R C, Dai X H, Zhao Y D.2017a. On improving CRISPR for editing plant genes: Ribozyme-mediated guide RNA production and fluorescence-based technology for isolating transgene-free mutants generated by CRISPR.Prog Mol Biol Transl, 149: 151-166.
[17]	He Y B, Zhang T, Yang N, Xu M L, Yan L, Wang L H, Wang R C, Zhao Y D.2017b. Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing.J Genet Genom, 44(9): 469-472.
[18]	He Y B, Zhu M, Wang L H, Wu J H, Wang Q Y, Wang R C, Zhao Y D.2018. Programmed self-elimination of the CRISPR/Cas9 construct greatly accelerates the isolation of edited and transgene-free rice plants.Mol Plant, 11(9): 1210-1213.
[19]	Hiei Y, Ohta S, Komari T, Kumashiro T.1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 6(2): 271-282.
[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, Liu D R.2018. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.Nature, 556: 57-63.
[21]	Hua K, Tao X P, Yuan F T, Wang D, Zhu J K.2018. Precise A∙T to G∙C base editing in the rice genome.Mol Plant, 11(4): 627-630.
[22]	Huang Y H, Su J Z, Lei Y, Brunetti L, Gundry M C, Zhang X T, Jeong M, Li W, Goodell M A.2017. DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A.Genome Biol, 18(1): 176.
[23]	Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J A, Charpentier E.2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science, 337: 816-821.
[24]	Kleinstiver B P, Prew M S, Tsai S Q, Topkar V V, Nguyen N T, Zheng Z, Gonzales A P, Li Z, Peterson R T, Yeh J R, Aryee M J, Joung J K.2015. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.Nature, 523: 481-485.
[25]	Komor A C, Kim Y B, Packer M S, Zuris J A, Liu D R.2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature, 533: 420-424.
[26]	Konermann S, Brigham M D, Trevino A E, Joung J, Abudayyeh O O, Barcena C, Hsu P D, Habib N, Gootenberg J S, Nishimasu H, Nureki O, Zhang F.2015. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.Nature, 517: 583-588.
[27]	Lei Y, Lu L, Liu H Y, Li S, Xing F, Chen L L.2014. CRISPR-P: A web tool for synthetic single-guide RNA design of CRISPR- system in plants.Mol Plant, 7(9): 1494-1496.
[28]	Li J Y, Sun Y W, Du J L, Zhao Y D, Xia L Q.2017. Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system.Mol Plant, 10(3): 526-529.
[29]	Li J Y, Zhang X, Sun Y W, Zhang J H, Du W M, Guo X P, Li S Y, Zhao Y D, Xia L Q.2018. Efficient allelic replacement in rice by gene editing: A case study of the NRT1.1B gene. J Integr Plant Biol, 60(7): 536-540.
[30]	Li P J, Wang Y H, Qian Q, Fu Z M, Wang M, Zeng D L, Li B H, Wang X J, Li J Y.2007. LAZY1 controls rice shoot gravitropism through regulating polar auxin transport.Cell Res, 17(5): 402-410.
[31]	Li S Y, Li J Y, Zhang J H, Du W M, Fu J D, Sutar S, Zhao Y D, Xia L Q.2018a. Synthesis-dependent repair of Cpf1-induced double-strand DNA breaks enables targeted gene replacement in rice.J Exp Bot, 69(20): 4715-4721.
[32]	Li S Y, Zhang X, Wang W S, Guo X P, Wu Z C, Du W M, Zhao Y D, Xia L Q.2018b. Expanding the scope of CRISPR/Cpf1- mediated genome editing in rice.Mol Plant, 11(7): 995-998.
[33]	Li Z X, Zhang D D, Xiong X Y, Yan B Y, Xie W, Sheen J, Li J F.2017. A potent Cas9-derived gene activator for plant and mammalian cells.Nat Plants, 3: 930-936.
[34]	Liu X S, Wu H, Ji X, Stelzer Y, Wu X B, Czauderna S, Shu J, Dadon D, Young R A, Jaenisch R.2016. Editing DNA methylation in the mammalian genome.Cell, 167(1): 233-247.
[35]	Lu Y M, Zhu J K.2017. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system.Mol Plant, 10(3): 523-525.
[36]	Mali P, Yang L H, Esvelt K M, Aach J, Guell M, DiCarlo J E, Norville J E, Church G M.2013. RNA-guided human genome engineering via Cas9.Science, 339: 823-826.
[37]	McElroy D, Zhang W G, Cao J, Wu R.1990. Isolation of an efficient actin promoter for use in rice transformation. Plant Cell, 2(2): 163-171.
[38]	Murovec J, Pirc Z, Yang B.2017. New variants of CRISPR RNA-guided genome editing enzymes.Plant Biotechnol J, 15(8): 917-926.
[39]	Ren B, Yan F, Kuang Y J, Li N, Zhang D W, Zhou X P, Lin H H, Zhou H B.2018. Improved base editor for efficiently inducing genetic variations in rice with CRISPR/Cas9-guided hyperactive hAID mutant. Mol Plant, 11(4): 623-626.
[40]	Stepper P, Kungulovski G, Jurkowska R Z, Chandra T, Krueger F, Reinhardt R, Reik W, Jeltsch A, Jurkowski T P.2017. Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.Nucleic Acids Res, 45(4): 1703-1713.
[41]	Sun Y W, Zhang X, Wu C Y, He Y B, Ma Y Z, Hou H, Guo X P, Du W M, Zhao Y D, Xia L Q.2016. Engineering herbicide- resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase.Mol Plant, 9(4): 628-631.
[42]	Tak Y E, Kleinstiver B P, Nunez J K, Hsu J Y, Horng J E, Gong J Y, Weissman J S, Joung J K.2017. Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors.Nat Methods, 14(12): 1163-1166.
[43]	Tanenbaum M E, Gilbert L A, Qi L S, Weissman J S, Vale R D.2014. A protein-tagging system for signal amplification in gene expression and fluorescence imaging.Cell, 159(3): 635-646.
[44]	Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, Klasic M, Zoldos V.2016. Repurposing the CRISPR-Cas9 system for targeted DNA methylation.Nucleic Acids Res, 44(12): 5615-5628.
[45]	Wang M G, Lu Y M, Botella J R, Mao Y F, Hua K, Zhu J K.2017. Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR/Cas9 system.Mol Plant, 10(7): 1007-1010.
[46]	Wang Z H, Zou Y J, Li X Y, Zhang Q Y, Chen L T, Wu H, Su D H, Chen Y L, Guo J X, Luo D, Long Y M, Zhong Y M, Liu Y G.2006. Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing.Plant Cell, 18(3): 676-687.
[47]	Weinhold A, Kallenbach M, Baldwin I T.2013. Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants. BMC Plant Biol, 13: 99.
[48]	Wilkinson J E, Twell D, Lindsey K.1997. Activities of CaMV 35S and nos promoters in pollen: Implications for field release of transgenic plants.J Exp Bot, 48(2): 265-275.
[49]	Xie X R, Ma X L, Zhu Q L, Zeng D C, Li G S, Liu Y G.2017. CRISPR-GE: A convenient software toolkit for CRISPR-based genome editing.Mol Plant, 10(9): 1246-1249.
[50]	Yan F, Kuang Y J, Ren B, Wang J W, Zhang D W, Lin H H, Yang B, Zhou X P, Zhou H B.2018. Highly efficient A∙T to G∙C base editing by Cas9n-guided tRNA adenosine deaminase in rice.Mol Plant, 11(4): 631-634.
[51]	Yoshihara T, Iino M.2007. Identification of the gravitropism- related rice gene LAZY1 and elucidation of LAZY1-dependent and -independent gravity signaling pathways. Plant Cell Physiol, 48(5): 678-688.
[52]	Yu C C, Wang L L, Xu S L, Zeng Y F, He C L, Chen C, Huang W C, Zhu Y G, Hu J.2015. Mitochondrial ORFH79 is essential for drought and salt tolerance in rice.Plant Cell Physiol, 56(11): 2248-2258.
[53]	Zetsche B, Gootenberg J S, Abudayyeh O O, Slaymaker I M, Makarova K S, Essletzbichler P, Volz S E, Joung J, van der Oost J, Regev A, Koonin E V, Zhang F.2015. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell, 163(3): 759-771.
[54]	Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro E M, Winblad N, Choudhury S R, Abudayyeh O O, Gootenberg J S, Wu W Y, Scott D A, Severinov K, van der Oost J, Zhang F.2016. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array.Nat Biotechnol, 35: 31-34.
[55]	Zhong Z H, Zhang Y X, You Q, Tang X, Ren Q R, Liu S S, Yang L J, Wang Y, Liu X P, Liu B L, Zhang T, Zheng X L, Le Y, Zhang Y, Qi Y P.2018. Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites.Mol Plant, 11(7): 999-1002.
[56]	Zhou Y X, Wang P, Tian F, Gao G, Huang L, Wei W S, Xie X S.2017. Painting a specific chromosome with CRISPR/Cas9 for live-cell imaging.Cell Res, 27: 298-301.

T₀ plant	Genotype of T₁ plant	Sequence analysis	Segregation
WT	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG
#1	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (7/7)
#1	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (7/7)
#11	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	10.00% (1/10)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	10.00% (1/10)
	BI, -10/-C	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	70.00% (7/10)
	BI, -10/-C	TCGAGTCGCGCCCGGAGTA-CTGCAGGCGCCGCGGG	70.00% (7/10)
	HO, -10/-10	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	20.00% (2/10)
	HO, -10/-10	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	20.00% (2/10)
#16	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	75.00% (6/8)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	75.00% (6/8)
	HE, WT/-CC	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	25.00% (2/8)
	HE, WT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	25.00% (2/8)
#24	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (4/16)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (4/16)
	HO, +T/+T	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	25.00% (4/16)
	HO, +T/+T	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	25.00% (4/16)
	BI, +T/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	43.75% (7/16)
	BI, +T/-C	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	43.75% (7/16)
	BI, -TACCT/-C	TCGAGTCGCGCCCGGAG-----GCAGGCGCCGCGGG	6.25% (1/16)
	BI, -TACCT/-C	TCGAGTCGCGCCCGGAGTA-CTGCAGGCGCCGCGGG	6.25% (1/16)
#27	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (18/18)
#27	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (18/18)
#29	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	13.33% (2/15)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	13.33% (2/15)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	26.67% (4/15)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	26.67% (4/15)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	60.00% (9/15)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	60.00% (9/15)
#32	HO, -ACCT/-ACCT	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	46.15% (6/13)
	HO, -ACCT/-ACCT	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	46.15% (6/13)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	53.85% (7/13)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	53.85% (7/13)
#36	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	14.29% (1/7)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	57.14% (4/7)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	57.14% (4/7)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	14.29% (1/7)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
#42	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (2/8)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (2/8)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	75.00% (6/8)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	75.00% (6/8)
#52	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (10/10)
#52	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (10/10)
#59	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	42.86% (6/14)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	42.86% (6/14)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	7.14% (1/14)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	7.14% (1/14)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	50.00% (7/14)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	50.00% (7/14)
WT, Wild-type Zhonghua 11 plant; HO, Homozygous mutation; HE, Heterozygous mutation; BI, Bi-allelic mutation. The underlined ‘AGG’ refers to the protospacer adjacent motif site required for Cas9 cleavage. ‘-’ refers to one base pair deletion. Lowercase letter ‘t’ in red refers to an insertion of a ‘T’. Number of plants for the genotype and the total number of the T₁ plants from each T₀ plant analyzed are shown in the parenthesis.

T₀ plant	Genotype of T₁ plant	Sequence analysis	Segregation
WT	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG
#1	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (7/7)
#1	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (7/7)
#11	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	10.00% (1/10)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	10.00% (1/10)
	BI, -10/-C	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	70.00% (7/10)
	BI, -10/-C	TCGAGTCGCGCCCGGAGTA-CTGCAGGCGCCGCGGG	70.00% (7/10)
	HO, -10/-10	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	20.00% (2/10)
	HO, -10/-10	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	20.00% (2/10)
#16	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	75.00% (6/8)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	75.00% (6/8)
	HE, WT/-CC	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	25.00% (2/8)
	HE, WT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	25.00% (2/8)
#24	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (4/16)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (4/16)
	HO, +T/+T	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	25.00% (4/16)
	HO, +T/+T	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	25.00% (4/16)
	BI, +T/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	43.75% (7/16)
	BI, +T/-C	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	43.75% (7/16)
	BI, -TACCT/-C	TCGAGTCGCGCCCGGAG-----GCAGGCGCCGCGGG	6.25% (1/16)
	BI, -TACCT/-C	TCGAGTCGCGCCCGGAGTA-CTGCAGGCGCCGCGGG	6.25% (1/16)
#27	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (18/18)
#27	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (18/18)
#29	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	13.33% (2/15)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	13.33% (2/15)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	26.67% (4/15)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	26.67% (4/15)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	60.00% (9/15)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	60.00% (9/15)
#32	HO, -ACCT/-ACCT	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	46.15% (6/13)
	HO, -ACCT/-ACCT	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	46.15% (6/13)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	53.85% (7/13)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	53.85% (7/13)
#36	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	14.29% (1/7)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	57.14% (4/7)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	57.14% (4/7)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	14.29% (1/7)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
#42	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (2/8)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (2/8)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	75.00% (6/8)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	75.00% (6/8)
#52	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (10/10)
#52	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (10/10)
#59	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	42.86% (6/14)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	42.86% (6/14)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	7.14% (1/14)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	7.14% (1/14)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	50.00% (7/14)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	50.00% (7/14)
WT, Wild-type Zhonghua 11 plant; HO, Homozygous mutation; HE, Heterozygous mutation; BI, Bi-allelic mutation. The underlined ‘AGG’ refers to the protospacer adjacent motif site required for Cas9 cleavage. ‘-’ refers to one base pair deletion. Lowercase letter ‘t’ in red refers to an insertion of a ‘T’. Number of plants for the genotype and the total number of the T₁ plants from each T₀ plant analyzed are shown in the parenthesis.

T₀ plant	Genotype of T₁ plant	Sequence analysis	Segregation
WT	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG
#3	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (22/22)
#3	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (22/22)
#4	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	100.00% (9/9)
#4	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	100.00% (9/9)
#5	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	16.67% (3/18)
	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	16.67% (3/18)
	HO, -CCT/-CCT	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	33.33% (6/18)
	HO, -CCT/-CCT	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	33.33% (6/18)
	BI, -CCT/-T	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	50.00% (9/18)
	BI, -CCT/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	50.00% (9/18)
#17	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	HO, +C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	11.11% (1/9)
	HO, +C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	11.11% (1/9)
	BI, -C/+C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	BI, -C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	33.33% (3/9)
	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	22.22% (2/9)
	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	22.22% (2/9)
#22	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	35.29% (6/17)
	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	35.29% (6/17)
	HO, +G/+G	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	23.53% (4/17)
	HO, +G/+G	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	23.53% (4/17)
	BI, +G/-T	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	41.18% (7/17)
	BI, +G/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	41.18% (7/17)
Protospacer adjacent motif site AGG is underlined. Zhonghua 11 plants were used as wild-type (WT). ‘+’ refers to base pair insertion. ‘-’ refers to base pair deletion. Lowercase letter ‘c’ and ‘g’ in red refer to insertions of ‘C’ and ‘G’, respectively. Number of plants for the genotype and the total number of the T₁ plants from each T₀ plant analyzed are shown in the parenthesis.

T₀ plant	Genotype of T₁ plant	Sequence analysis	Segregation
WT	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG
#3	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (22/22)
#3	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (22/22)
#4	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	100.00% (9/9)
#4	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	100.00% (9/9)
#5	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	16.67% (3/18)
	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	16.67% (3/18)
	HO, -CCT/-CCT	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	33.33% (6/18)
	HO, -CCT/-CCT	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	33.33% (6/18)
	BI, -CCT/-T	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	50.00% (9/18)
	BI, -CCT/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	50.00% (9/18)
#17	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	HO, +C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	11.11% (1/9)
	HO, +C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	11.11% (1/9)
	BI, -C/+C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	BI, -C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	33.33% (3/9)
	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	22.22% (2/9)
	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	22.22% (2/9)
#22	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	35.29% (6/17)
	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	35.29% (6/17)
	HO, +G/+G	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	23.53% (4/17)
	HO, +G/+G	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	23.53% (4/17)
	BI, +G/-T	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	41.18% (7/17)
	BI, +G/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	41.18% (7/17)
Protospacer adjacent motif site AGG is underlined. Zhonghua 11 plants were used as wild-type (WT). ‘+’ refers to base pair insertion. ‘-’ refers to base pair deletion. Lowercase letter ‘c’ and ‘g’ in red refer to insertions of ‘C’ and ‘G’, respectively. Number of plants for the genotype and the total number of the T₁ plants from each T₀ plant analyzed are shown in the parenthesis.

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 3

参考文献 56

相关文章 6

编辑推荐

Metrics

[1]	LIU Tingting, ZOU Jinpeng, YANG Xi, WANG Kejian, RAO Yuchun, WANG Chun. Development and Application of Prime Editing in Plants[J]. Rice Science, 2023, 30(6): 3-.
[2]	. [J]. Rice Science, 2019, 26(2): 118-124.
[3]	. [J]. Rice Science, 2019, 26(2): 77-87.
[4]	. [J]. Rice Science, 2019, 26(2): 88-97.
[5]	. [J]. Rice Science, 2019, 26(2): 98-108.
[6]	. [J]. Rice Science, 2019, 26(2): 69-761.