CRISPR/Cas9-Mediated Adenine Base Editing in Rice Genome

doi:10.1016/j.rsci.2018.07.002

Abstract

Hao Li, Ruiying Qin, Xiaoshuang Liu, Shengxiang Liao, Rongfang Xu, Jianbo Yang, Pengcheng Wei. CRISPR/Cas9-Mediated Adenine Base Editing in Rice Genome[J]. Rice Science, 2019, 26(2): 125-128.

Fig. 1. Targeted adenine base editing (ABE) in rice genome.A, Schematic diagrams of rice codon-optimized tools pHUN411-ABE and pHUN411-ABE-sg2.0. B, Schematic illustration of the target site in the Wx gene. The protospacer adjacent motif (PAM) and sgRNA sequences are underlined. The desired point mutation of Wx-mq is indicated in red. C, Representative Sanger sequencing chromatograms of the ABE-edited Wx allele. Arrows mark the T to C substitutions. The protospacer sequence is underlined. D, The base editing efficiency of the different sgRNAs and different targets. E, Schematic illustration of the target site in the GL2 and OsGRF2 genes by the GL2-sg protospacer. The PAM and sgRNA sequences are underlined. The binding sequence of the miR396 is indicated in red. The asterisks show the mutation sites in the wild-type GL2 allele with large grain size. F, Representative Sanger sequencing chromatograms of the ABE-edited GL2 allele. RB, Right border; LB, Left border; NTL, Number of transgenic line; NEL, Number of edited line; BEE, Base editing efficiency.

Supplemental Table 1 The primers used in this study.

Element	Primer (5′-3′)	Function
ecTadA-XTEN-TadA710*	F: TTACGCCAAGCTGCCCTTGGCGGCCGCATGAGCGAAGTGGA	ABE710 vector construction
ecTadA-XTEN-TadA710*	R: GCGGGTCCTCCGGTGGTTCCGACAAGAAGTACTCCATCGG	ABE710 vector construction
nSpCas9(D10A)	F: GCGGGTCCTCCGGTGGTTCCGACAAGAAGTACTCCATCGGCCTCGCTATCGGCACCAAT	ABE710 vector construction
nSpCas9(D10A)	R: GCGAATTGAAGCTGCCCTTGGAGCTCTCACACCTTTCTTTTTTTTTTAGGAGAACCACCAGA	ABE710 vector construction
Wx-sg	F: GGCATTGTAATCAACTCCAGTGTC	sgRNA construction
Wx-sg	R: AAACGACACTGGAGTTGATTACAA	sgRNA construction
GL2-sg	F: GGCATTCTTGAACGGTTGCGGCCG	sgRNA construction
GL2-sg	R: AAACCGGCCGCAACCGTTCAAGAA	sgRNA construction
Wx	F: ACTGGTGATTTCAGGTTTGGGG	HRM
	R: TGACCTGGCAAAGAAGGCTGA	HRM
	F: GAGGTTTTTCCATTGCTACAAG	PCR sequencing
	R: GAAGTATGGGTTGTTGTTGAGG	PCR sequencing
GL2	F: ACGGACGGCAAGAAATGGCG	HRM
	R: CGCCGCAGAACCGACAACAG	HRM
	F: CGGTTGCTACGATGTGCCTGTT	PCR sequencing
	R: AGAACCCAACGCCGAGCCAAAT	PCR sequencing
OsGRF3	F: AAGAAGTGGCGGTGCTCCAAGG	HRM
	R: GAGTGGTTCTGGAAGGCGGTGG	HRM
	F: TGGCTGTAAAGCGTTCCATTCC	PCR sequencing
	R: GAGTATTAGACCCCAAGGCGAA	PCR sequencing
OsGRF5	F: CCGCACCGACGGCAAGAAGT	HRM (off-target detection)
	R: AGGGACAGACCGTGGGTTTTAG	HRM (off-target detection)
	F: GGATTCAATGTGCCTGTGCT	PCR sequencing (off target detection)
	R: GTCAATGAGATTAGAGGGGA	PCR sequencing (off target detection)

Supplemental Table 2 Identification of targeted wx mutants induced by ABE tools in transgenic rice

Line	Target gene	Zygosity*	Genotype#
Wx-6	Wx	Heterozygous	WT/T₆toC
Wx-15	Wx	Chimeric	WT/ T₅toC/ T₆toC
Wx-sg2.0-2	Wx	Chimeric	WT/T₅toC/T₆toC
Wx-sg2.0-7	Wx	Heterozygous	WT/T₅toC
Wx-sg2.0-15	Wx	Chimeric	WT/T₆toC/T₉toC
Wx-sg2.0-21	Wx	Heterozygous	WT/T₅T₆toCC
Wx-sg2.0-22	Wx	Heterozygous	WT/T₆toC
*, The zygote of the mutant is putatively determined by the results of PCR sequencing and/or clone sequencing. ^#, WT, wild-type; T_xtoC, the T at position x is mutated to C.

Supplemental Table 3 Identification of targeted gl2 and/or osgrf3 mutants induced by ABE tools in transgenic rice.

Line	Target gene	Zygosity*	Genotype^#
GL2-9	GL2	Heterozygous	WT/T₇toC
GL2-9	OsGRF3	Heterozygous	WT/T₇toC
GL2-17	GL2	Heterozygous	WT/T₇toC
GL2-17	OsGRF3	WT	WT
GL2-22	GL2	Heterozygous	WT/T₈toC
GL2-22	OsGRF3	Heterozygous	WT/T₇toC
GL2-33	GL2	Heterozygous	WT/T₇toC
GL2-33	OsGRF3	Heterozygous	WT/T₇toC
GL2-sg2.0-6	GL2	Chimeric	WT/ T₇toC/ T₈toC
GL2-sg2.0-6	OsGRF3	Heterozygous	WT/ T₇toC
GL2-sg2.0-11	GL2	WT	WT
GL2-sg2.0-11	OsGRF3	Heterozygous	WT/ T₇toC
GL2-sg2.0-14	GL2	WT	WT
GL2-sg2.0-14	OsGRF3	Chimeric	WT/ T₇toC/ T₇T₈toCC
GL2-sg2.0-23	GL2	Heterozygous	WT/ T₈toC
GL2-sg2.0-23	OsGRF3	Heterozygous	WT/ T₇T₈toCC
GL2-sg2.0-30	GL2	Heterozygous	WT/ T₈toC
GL2-sg2.0-30	OsGRF3	Heterozygous	WT/ T₇toC
GL2-sg2.0-34	GL2	Heterozygous	WT/ T₇toC
GL2-sg2.0-34	OsGRF3	WT	WT
*, The zygote of the mutant is putatively determined by the results of PCR-sequencing and/or clone-sequencing. ^#, WT, wild-type; T_xtoC, the T at position x is mutated to C.

Supplemental Table 4 Detection of the putative off-target effects of the rice ABE tools on the high-similarity site of the GL2-sg protospacer.

Off-target locus	Transgenic plant	Number of detected plants*		Number of off-target mutants
Off-target locus	Transgenic plant	HRM	Sequencing	HRM	Sequencing
OsGRF5 miR396 binding site	ABE-GL-sg	35	24	0	0
OsGRF5 miR396 binding site	ABE-sg2.0-GL-sg	30	24	0	0

Fig. 1. Sequence of rice codon-optimized ABE7.10.The sequence of wild type ecTadA, linker, TadA*7.10, nCas9(D10A) and NLS was labeled by blue, orange, red, black and cyan respectively.

Fig. 2 Genotyping of some targeted wx mutants.The mutations in the target region are indicated in red. The genotype of the wx targeted mutant was analyzed by PCR sequencing. The numbers located far right indicate the ratio of the number of clones with the corresponding genotype to the number of total sequenced clones. WT, wild-type; Tx to C, the T at position x is mutated to C.

Fig. 3 The sequence of the extended sgRNA scaffold.The structures of the sgRNA-scaffold-2.0 sequence were generated with RNAfold and visualized using VARNA (Darty et al., 2009). The adapted and extended RNA is shown in red.

Fig. 4 Sanger sequencing chromatograms of the OsGRF3 target region of representative ABE-Gl2-sg edited lines.The targeted T to C mutation(s) is indicated by arrow(s). The target sequence is underlined.

Fig. 5 Genotyping of some transgenic plants targeted by ABE-GL2-sg.The mutations within the target region are indicated in red. The genotype of the GL2 and OsGRF3 target regions of the mutant was analyzed by PCR sequencing.

Fig. 6 Alignment of the miR396 binding region in the rice growth-regulating factor family.The GL2-sg shows 100% sequence identity with GL2 and OsGRF3, whereas there is one base mismatch with that of OsGRF5.

Fig. 7 The mutation of protein sequence resulting from the adenine base editor-induced editing.The amino acid of the target region of Wx (A) and GL2/OsGRF3 (B) was indicated. The PAM and protospacer sequence was labeled by blue and orange, respectively.

References 16

[1]	Che R H, Tong H N, Shi B H, Liu Y Q, Fang S R, Liu D P, Xiao Y H, Hu B, Liu L C, Wang H R, Zhao M F, Chu C C.2015. Control of grain size and rice yield by GL2-mediated brassinosteroid responses.Nat Plants, 2: 15195.
[2]	Dang Y, Jia G X, Choi J, Ma H M, Anaya E, Ye C T, Shankar P, Wu H Q.2015. Optimizing sgRNA structure to improve CRISPR- Cas9 knockout efficiency.Genome Biol, 16: 280.
[3]	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 A∙T to G∙C in genomic DNA without DNA cleavage.Nature, 551: 464-471.
[4]	Hu X X, Meng X B, Liu Q, Li J Y, Wang K J.2018. Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice.Plant Biotechnol J, 16(1): 292-297.
[5]	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.
[6]	Koblan L W, Doman J L, Wilson C, Levy J M, Tay T, Newby G A, Maianti J P, Raguram A, Liu D R.2018. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction.Nat Biotechnol, doi: 10.1038/nbt.4172.
[7]	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.
[8]	Li C, Zong Y, Wang Y P, Jin S, Zhang D B, Song Q N, Zhang R, Gao C X.2018. Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion.Genome Biol, 19: 59.
[9]	Liu H H, Guo S Y, Xu Y Y, Li C H, Zhang Z Y, Zhang D J, Xu S J, Zhang C, Chong K.2014. OsmiR396d-regulatedOsGRFs function in floral organogenesis in rice through binding to their targets OsJMJ706 and OsCR4. Plant Physiol, 165(1): 160-174.
[10]	Lu Y, 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.
[11]	Miki D, Zhang W X, Zeng W J, Feng Z Y, Zhu J K.2018. CRISPR/ Cas9-mediated gene targeting inArabidopsis using sequential transformation. Nat Commun, 9: 1967.
[12]	Sato H, Suzuki Y, Sakai M, Imbe T.2002. Molecular characterization ofWx-mq, a novel mutant gene for low-amylose content in endosperm of rice(Oryza sativa L.). Jpn J Breeding, 52(2): 131-135
[13]	Xing H L, Dong L, Wang Z P, Zhang H Y, Han C Y, Liu B, Wang X C, Chen Q J.2014. A CRISPR/Cas9 toolkit for multiplex genome editing in plants.BMC Plant Biol, 14: 327.
[14]	Xu R F, Yang Y C, Qin R Y, Li H, Qiu C H, Li L, Wei P C, Yang J B.2016. Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice.J Genet Genom, 43(8): 529-532.
[15]	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.
[16]	Zong Y, Wang Y P, Li C, Zhang R, Chen K L, Ran Y D, Qiu J L, Wang D W, Gao C.2017. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion.Nat Biotechnol, 35(5): 438-440.