supplemental information precise a·t to g·c base editing ......supplemental figure 3. multiplex...

Supplemental information

Precise A·T to G·C base editing in the rice genome

Kai Hua, Xiaoping Tao, Fengtong Yuan, Dong Wang, Jian-Kang Zhu

Contents

Supplemental Figure 1. TA cloning results of two base editing lines (SG1-7 and

SG1-15) containing pRABEsp-OsU6 with sgRNA1.

Supplemental Figure 2. pRABEsp-OsU6 can be used for multiplex base editing in

rice.

Supplemental Figure 3. Multiplex base editing at OsSPL16 and OsSPL18 by

pRABEsa-OsU6sa.

Supplemental Table 1. Base editing activity window for ABE-P1.

Supplemental Table 2. Base editing frequency at potential off-target sites of

sgRNA1.

Supplemental Table 3. Base editing activity window for ABE-P2.


sgRNA4.

Supplemental Table 5. Primers used in this study.

Supplemental Materials and Methods

Supplemental References

Supplemental Figure 1. TA cloning results of two base editing lines (SG1-7 and

SG1-15) containing pRABEsp-OsU6 with sgRNA1. Twenty clones from each line

were randomly selected for sequencing. Representative sequence chromatograms for

each genotype are shown. Arrows point to the positions with edited base.

Supplemental Figure 2. pRABEsp-OsU6 can be used for multiplex base editing

in rice. (A) sgRNA3 was designed for simultaneous editing of three genes in the rice

genome. The OsmiR156 binding sites in the three genes are highlighted in red. (B)

Representative sequence chromatograms at the target sites are shown. Note that in

lines SG3-11 and SG3-12, the target sites in OsSPL16 and OsSPL18 were

simultaneously edited. Arrows point to the positions with edited base.

Supplemental Figure 3. Multiplex base editing at OsSPL16 and OsSPL18 by

pRABEsa-OsU6sa. (A) The sgRNA5 was designed for simultaneous editing of

OsSPL16 and OsSPL18. (B) Representative sequence chromatograms for the two

target sites in three transgenic lines, SG5-7, SG5-18, and SG5-44. Both target genes

were edited in the three lines. Arrows point to the positions with edited base.

Supplemental Table 1. Base editing activity window for ABE-P1

sgRNA Target Line number Baseediting position Editing form

sgRNA1 OsSPL14 SG1-11, SG1-21 5 T-C conversion

SG1-10, SG1-15 5,7 T-C conversion

SG1-7, SG1-23 5,10 T-C conversion

sgRNA2 SLR1 SG2-4, SG2-18, SG2-19, SG2-26, SG2-36 6 T-C conversion

sgRNA3 OsSPL16 SG3-3, SG3-11, SG3-12, SG3-13 7 T-C conversion

OsSPL18 SG3-11, SG3-12 7 T-C conversion

SG3-15 7,9 T-C conversion

SG3-19 5,7 T-C conversion

LOC_Os02g24720 SG3-6 5 T-C conversion

Note: Base editing position was counted from the PAM-distal end, scoring the PAM

as positions 21-23.

Supplementary Table 2. Base editing frequency at potential off-target sites of

sgRNA1

Site Chromosome Position Guide-PAM sequence Mismatch

numbers

Editing

efficiency

On target 8 25275163 AGAGAGAGCACAGCTCGAGTCGG 0 26%

Off-target 1 8 18918918 AGAGAGAGCACAGCTgGAGTCGG 1 0

Off-target 2 1 24179644 tGAtcGgGCACAGCTCGcGTCGG 5 0

Off-target 3 3 36137080 AGAaAGAGCAtgGgTCGAGTCGG 4 0

Off-target 4 3 8596704 AGAGtGAGCACAGCggGAGaCGG 4 0

Off-target 5 4 23025825 gGAGAGcGCgCgGCTCGAGgCGG 5 0

Off-target 6 5 10284901 AGAGtGtGCAgAGtTCGAGTCGG 4 0

Off-target 7 7 19030423 AGAGAGAGCtCgGCTCGgcTCGG 4 0

Off-target 8 10 1012640 AGAGAGAtCtCAGaTCGAGgCGG 4 0

Off-target 9 7 20804240 AGAcAGAGCACAGCaaGAaTCGG 4 0

Note: Nucleotides of the PAM sequence are written in bold, and the mismatch bases

in potential off-targets are shown in lowercase.

Supplementary Table 3. Base editing activity window for ABE-P2

sgRNA Target Line number Base editing position Editing form

sgRNA4 OsSPL14 SG4-6, SG4-11, SG4-16 8 T-C conversion

SG4-2, SG4-5, SG4-17, SG4-19 10 T-C conversion

SG4-4 6, 10 T-C conversion

SG4-1, SG4-24, SG4-27 8, 10 T-C conversion

SG4-7, SG4-10 6, 8, 10 T-C conversion


OsSPL17 SG4-2, SG4-7 8 T-C conversion

SG4-8 10 T-C conversion

SG4-12, SG4-20 12 T-C conversion

SG4-1, SG4-5, SG4-6, SG4-10, SG4-16,

SG4-18, SG4-24, SG4-26

8, 10 T-C conversion

SG4-11, SG4-19 8, 10, 12 T-C conversion

SG4-27 8, 10,14 T-C conversion

SG-3 6, 10, 12, 14 T-C conversion

SG17, SG4-25 8, 10, 12, 14 T-C conversion

sgRNA5 OsSPL16 SG5-18, SG5-30, SG5-45 12 T-C conversion

SG5-7, SG5-23, SG5-32, SG5-33, SG5-44 14 T-C conversion

OsSPL18 SG5-18, SG5-21, SG5-29, SG5-37, SG5-38,

SG5-40, SG5-44, SG5-45

10 T-C conversion


SG5-23 8, 10, 12 T-C conversion

SG5-12 10, 12, 14 T-C conversion

Note: Base editing position was counted from the PAM-distal end, scoring the PAM

as positions 22-27.


sgRNA4

Site Chromosome Position Guide-PAM sequence Mismatch

numbers

Editing

efficiency

On target 1 8 25275156 ACAGAAGAGAGAGAGCACAGCTCGAGT 0 45.2%

On target 2 9 18918911 ACAGAAGAGAGAGAGCACAGCTGGAGT 0 61.3%

Off-target 1 9 19647839 ACAGAAGAGAGAGAGCACAat CGGAGT 2 0

Off-target 2 11 17631827 ACAGAAGAGAGAGAGCACAct CCGGGT 2 0

Off-target 3 8 26505555 ACAGAAGAGAGAGAGCACAct CCGGGT 2 0

Off-target 5 3 36137083 ACgGAAGAGAaAGAGCAtgGg TCGAGT 5 0

Note: Nucleotides of PAM sequence are written in bold, and the mismatch bases in

potential off-targets are shown in lowercase.

Supplemental Table 5. Primers used in this study

Primer name Primer sequence 5’-3’ Purpose

sgRNA1-F TGTGAGAGAGAGCACAGCTCGAGT sgRNA1vector construction

sgRNA1-R AAACACTCGAGCTGTGCTCTCTCT

sgRNA2-F TGTGAGTGCACGGTGTCCGTGGCC sgRNA2 vector construction

sgRNA2-R AAACGGCCACGGACACCGTGCACT

sgRNA3-F TGTGCAGAAGAGAGAGAGCACAAT sgRNA3 vector construction

sgRNA3-R AAACATTGTGCTCTCTCTCTTCTG

sgRNA4-F TGTGTGACAGAAGAGAGAGAGCACAGC sgRNA4 vector construction

sgRNA4-R AAACGCTGTGCTCTCTCTCTTCTGTCA

sgRNA5-F TGTGTGACAGAAGAGAGAGAGCACAAT sgRNA5 vector construction

sgRNA5-R AAACATTGTGCTCTCTCTCTTCTGTCA

SPL14-seq-F AGGGTTCCAAGCAGCGTAAGGA Genotyping the target sites in OsSPL14

SPL14-seq-R TGGTGCTGGGGCTGGACCGTTC

SLR-seq-F GCGCAATTATTACTAGCTATAGC Genotyping the target site in SLR1

SLR-seq-R AGCCGTCGCCACCACCGGTAAGG

SPL16-seq-F CAGCTGCTGCTGCTGCTTCAGTGTG Genotyping the target sites in OsSPL16

SPL16-seq-R ACATCCCATTGTAGTTCATCTCATTG

02G-seq-F GCCTGCAGGCGGAGGAGTGG Genotyping the target site in LOC_Os02g24720

02G-seq-R AGTTCATCTCATTGTCATTGGA

SPL17-seq-F GGACCTCGTTCAGCACAACCC Genotyping the target site in OsSPL17

SPL17-seq-R GGGTTCCAAGCAGTGTGAGGGA

SPL18-seq-F TGGGATCATCAAATCCGAGGAG Genotyping the target sites in OsSPL18

SPL18-seq-R CTGTCCATGCTCGGGCAGGCG

sgRNA1M1-F GGGACCTCGTTCAGCACAACCCC Off target site 1 detection for sgRNA1

sgRNA1M1-R GCAGGTCCAGAAGCTTTGTGGTA

sgRNA1M2-F TTGCTCCCTAAACAACTCCCAG Off target site 2 detection for sgRNA1

sgRNA1M2-R CCTGGTGCAGAGAGTACAAATG

sgRNA1M3-F TGCAATGCATCTACTCCCTCAG Off target site 3 detection for sgRNA1

sgRNA1M3-R GATCACACTAGCGACAGCGAGC

sgRNA1M4-F ACGAGAGCTTCGACTGAACGCA Off target site 4 detection for sgRNA1

sgRNA1M4-R AATCTGCCGCGTGTCAGCAGAG

sgRNA1M5-F TGCAGGTGGTCGGCGATCGCG Off target site 5 detection for sgRNA1

sgRNA1M5-R AGCGCGCGCAGCTTGGCCT

sgRNA1M6-F GATCACAACTGCTCGTAAGCTA Off target site 6 detection for sgRNA1

sgRNA1M6-R ATATGTCCTTATCGGACACGAC

sgRNA1M7-F CCAAACTCTATCTTGAGCCTTC Off target site 7 detection for sgRNA1

sgRNA1M7-R GAGTTCGACGAGGGAGAGAGA

sgRNA1M8-F ATATTCATAATCCCCCTAGAA Off target site 8 detection for sgRNA1

sgRNA1M8-R CCCCAACGCTTCGCGAATCGCA

sgRNA1M9-F CAGAGCCTGGACGGGTTCTAC Off target site 9 detection for sgRNA1

sgRNA1M9-R ACAGTACAGCAAATCGCACGT

sgRNA4M1-F GCAACAAGATGTTCTCCTCCG Off target site 1 detection for sgRNA4

sgRNA4M1-R CTGCCGCCGAACTGCTGCTGC

sgRNA4M2-F ACACCTGCCAAGAGAATGGCA Off target site 2 detection for sgRNA4

sgRNA4M2-R CCGATAGCTGATCAGAACGC

sgRNA4M3-F TTGGCATCAGCAGCAGGCAGCA Off target site 3 detection for sgRNA4

sgRNA4M3-R GAACCAGGCTGAGCTGCCGCC

sgRNA4M4-F TATTATTCGTGCAATGCATCTA Off target site 4 detection for sgRNA4

sgRNA4M4-R ATGCGAGCGGTCTAACCGACGA

Materials and methods

Vector construction

The mammalian codon-optimized wild type E. coli tRNA adenine deaminase

ecTadA(wt) and its mutated form ecTadA*(7.10) with a 96 bp linker were synthesized

by GENEWIZ (Suzhou, China) as described in Gaudelli et al (Gaudelli et al., 2017).

Two AarI sites with appropriate overhangs were added at both ends of edTadA(wt)

and ecTadA*(7.10). The vector pCas9(OsU6), containing a Cas9 gene driven by the

maize ubiquitin promoter, was modified to construct adenine base editing vectors.

First, SpCas9 (D10A) nickase and SaCas9 (D10A) nickase flanked by two AarI sites

at 5’ terminal and VirD2 nuclear localization signal (NLS) at 3’ terminal were

amplified from pCas9(OsU6) and pX600 (Addgene, #61592) to replace the Cas9 gene

in the pCas9(OsU6) vector to form intermediate vectors pRSp-OsU6 and pRSa-OsU6,

respectively. Then, the edTadA(wt) and ecTadA*(7.10) with 96 bp linker were

inserted between two AarI sites of pRSp-OsU6 and pRSa-OsU6 by the Golden Gate

method, leading to pRABESp-OsU6 and pRABESa-OsU6. A fragment comprised of

the OsU6 promoter, two BsaI sites and the sgRNA scaffold matching SaCas9 was

obtained by overlap PCR. Then it was used to replace the OsU6-spsgRNA cassette in

pRABESp-OsU6 to form pRABESa-OsU6Sa. The backbone of the final vectors,

pRABESp-OsU6 and pRABESa-OsU6Sa, contains the hygromycin B

phosphotransferase (hpt) gene for transgene selection.

The 20 bp (for SpCas9) or 21 bp (for SaCas9) sgRNA target sequences were

synthesized and annealed on a PCR machine. The annealed oligo adaptors were

inserted into the BsaI digested pRABESp-OsU6 and pRABESa-OsU6Sa vectors. The

accuracy of vectors was confirmed by Sanger sequencing. Primers used for vector

construction are listed in Supplemental Table 5.

Rice transformation

All binary vectors were transformed into the A. tumefaciens strain EHA105 by the

freeze/thaw method. Transformation of embryogenic calli induced from mature seeds

of Nipponbare rice (Oryza sativa L. japonica. cv. Nipponbare) was performed as

described previously with minor modifications (Nishimura et al., 2007). Briefly, two

days after Agrobacterium infection, calli were transferred onto selection media for

one round of selection for two weeks. Then, the resistant calli were directly

transferred to regeneration media for shoot regeneration. After the shoots grew to 4-5

cm in length, the plantlets were transferred to MS media for root induction. Two

weeks later, the plantlets were transplanted to soil pots and grew in a greenhouse

under standard conditions (12-h light 28°C and 12-h darkness at 22°C).

Genotyping editing events

Genomic DNA was extracted from the leaves of all T0 transgenic lines. Each target

locus was amplified by PCR and the PCR products were purified for Sanger

sequencing. Some PCR sequencing results were further confirmed by TA cloning and

sequencing. Base editing ratio was calculated by scoring the number of plants with

base editing events divided by the total number of genotyped transgenic lines. Primers

for genotyping each target site are shown in Supplemental Table 5.

Off-target detection

Off-target sites prediction was done by the online tool CRISPR-GE (Xie et al., 2017).

Homologous sequences with up to 5 bp mismatches to target sites were listed as

potential off-target sites. The potential off-target sites were each amplified from base

edited lines for Sanger sequencing. Primers for off-target amplification are listed in

Supplemental Table 5.

Supplemental References

Gaudelli, N.M., Komor, A.C., Rees, H.A., Packer, M.S., Badran, A.H., Bryson, D.I.,

and Liu, D.R. (2017). Programmable base editing of A•T to G•C in genomic

DNA without DNA cleavage. Nature 551:464-471.

Nishimura, A., Aichi, I., and Matsuoka, M. (2007). A protocol for

Agrobacterium-mediated transformation in rice. Nat. Protoc. 1:2796-2802.

Xie, X., Ma, X., Zhu, Q., Zeng, D., Li, G., and Liu, Y.-G. (2017). CRISPR-GE: A

Convenient Software Toolkit for CRISPR-Based Genome Editing. Mol. Plant

10:1246-1249.

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