Development of high-efficiency plant genome editing toolkit and its use in

gene functional study

Yao-Guang Liu

刘耀光

South China Agricultural University

华南农业大学

Our objectives

I. Development of a robust, high-efficiency

CRISPR/Cas9 vector and software toolkit

for multiplex genome editing in plants

II. Use of the genome editing toolkit to study

gene function and genetic improvement in

plants

A plant CRISPR/Cas9 multiplex genome editing system

CRISPR/Cas9binary vectors(Ma et al. MP 2015)

sgRNA intermediate vectors

5’-GGCGCTCGCCAAGTTCGACAGGG-3’

5’-GCCGGCGCTCGCCAAGTTCGACA-3’(FP)3’-CGCGAGCGGTTCAAGCTGTCAAA-5’(RP)

Target sequence:

Target adaptor: 5’-GTTGTGTGTGCTTACAGCCATGGC-3’3’-ACACACGAATGTCGGTACCGCAAA-5’

5’-TGTGTGCTTACAGCCATGGCAGG-3’PAM PAMIrregular targetRegular target

19 bp 20 bp

D

Transcript (5’ part): 5’-GGCGCTCGCCAAGTTCGACAGTTT-- 5’-GTGTGTGCTTACAGCCATGGCGTTT--

Extra nucleotide

3’-NCCGCGAGCGGTTCAAGCTGTCCCN-5’Genomic binding site: 3’-NACACACGAATGTCGGTACCGTCCN-5’

NGGTCTCNNNNNN

NCCAGAGNNNNNN

BsaI

(1) Golden Gate ligation

ctcggact

ctgattctaaga

ctgagRNAT1

gRNAT2

gRNAT3

gact gRNA

T4

gagc(B-L )

(B1’) (B2’) (B3’) (B4’)

(B2) (B3) (B4)

Mlu IMlu I

U6b prU6a prU3 prU6c pr

(BL)E.coli Pr

LacZ cggtgcca

Spe I

(B-R )

Assembly of multiple sgRNA expression cassettes into a CRISPR/Cas9 binary vector by single round of cloning

BsaI(B-L)

BsaI(B-R)

pYLCRISPR/Cas9Pubi-H (16419 bp)

RB

Pu

bi

LB

2xP35S HPT/Bar

Cas9p

NLS

pBR322 oriKanR

NLS

pVS1 replicon

pYLCRISPR/Cas9Pubi-B (15889 bp)

T35S

Tnos

U#U#

U#

Ptac promoter F primer

Sp6 promoter primer

(2) Gibson Assembly

Sp6 universal primerT2

T3T4

T1U3

LacZ

Mlu I

Mlu I

GAS-L

Spe I

BsaI(B-L)

BsaI(B-R)

pYLCRISPR/Cas9Pubi-H (16419 bp)

RB

Pu

bi

LB

2xP35S HPT/Bar

Cas9p

NLS

pBR322 oriKanR

NLS

pVS1 replicon

pYLCRISPR/Cas9Pubi-B (15889 bp)

T35S

Tnos

Assembly of multiple sgRNA expression cassettes into a CRISPR/Cas9 binary vector by single round of cloning

Construction of CRISPR/Cas9 binary constructs for targeting multiple genes

Mlu I-digestion

Vector Linked sgRNA cassettes

Simultaneous editing of seven genes

Features of CRISPR/Cas9-based genome editing in rice

53.3

20.0

8.9

64.3

100

80

60

40

20

OsU6a(18, 140)

40.7

39.025.6

OsU6b(9, 59)

OsU6c(5, 39)

OsU3(14, 90)

46.2 54.9

Sum (46, 328)

10.3

90.0

1.781.4 82.1 82,2 85.4

Biallelic Homozygous Heterozygous

21.4

4.35.8

24.7

Edi

ting

rate

(%

)

59.8

26.2

2.5

51.5

16.8

8.9

100

80

60

40

20

30-50 (14, 101)

36.0

51-60(15, 105)

61-85(17, 122)

52.4

77.2

89.6 88.56.7

Biallelic Homozygous Heterozygous

GC%

Edi

ting

rate

(%)

Efficiencies of different U3 & U6 promotersEfficiencies of targets with different GC contents

No. targets No. events

A Web-based CRISPR-GE Toolkit（Web-site：http://skl.scau.edu.cn/)(Xie et al. MP 2017)

targetDesign/offTarget

Submission pageSelect PAM type (NGG, TTN, TTTN)

Select a reference genome (28 plant genomes and 5 animal genomes)

past a genomic sequence for editting

Or define a PAM type by user

Or input a gene locus

Result page

Potential off-target sites and scores

Goto primerDesign

Allelic types of targeted mutations in rice and other diploid organisms

GTCTGAATCTTTTTCACTaGCAGGttgct-3 Allele 1GTCTGAATCTTTTTCACTaGCAGGttgct-3 Allele 2GTCTGAATCTTTTTCACT-GCAGGttgct-3 Reference

Homozygous

mutation

Heterozygous/

biallelic

mutations

Chimeric

Mutations

(very few) Sequencing chromatogram with overlapped peaks

• Cloning of the PCR amplicons and sequencing multiple clones (>10) for each target site are time-consuming and high-cost !

• To resolve this problem, we developed a web-

based tool “DSDecode” and its updated version

“DSDecodeM”

Submission page of DSDecodeM

Past a wild-type reference sequence

Upload up to 20 sequencing files (ab1 file)

Results outputDownload results in a txt file

Worldwide decoded sequencing file number of targeted editing per month by DSDecode/DSDeceodeM

3661241

16141418

1174

1251

597 5111757

2508

2223

2247

2239

5003

5987

4645

7542

5870

3594 3768

4562

4747

57128268

13517

18588

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Use of the genome editing toolkit to

study gene function and genetic

improvement in plants

Strategies for targeted editing of genes

CRE: cis-regulatory element

Targeted editing in CDS of a gene

Targeted editing in one, two, or multiple CREs of a gene

*

Qualitative or quantitative variation of the trait

Quantitative variation of the trait

A

B

Cis-regulatory mutant alleles with altered expression level/pattern

Null-mutant alleles

Targeted editing in duplicated genes

Quantitative variation of

the trait

Gene dosage-reduced mutant alleles

*

A a-1

A1 A2 A1 a2

A1 A2 A2

CREsC

A

A

A

A

A-1

A-2

A-3

A-4

A-n

A1

a-2

Study on rice hybrid sterility by genome editing

栽培稻Cultivated rice

亚洲稻

非洲稻

粳稻

籼稻

（Oryza genus）

（Species）

（Subspecies）(Indica)

(Japonica)

(O. sativa)

(O. glaberrima)

野生稻Wild rice

Hybrid sterility (HS)

（male, female, male/female）

Cloned hybrid sterility genes in rice

Locus Sterility Gene structure Gene product Reference

Sa Male Two adjacent F-box, Long et al., 2008;genes SaF/SaM SUMO-ligase Xie et al., 2017a

S5 Female Three adjacent Aspartic Chen et al; 2008

genes protease & ? Yang et al., 2012

S27/S28 Male Duplicated Ribosomal protein Yamagata et al., 2010

genes DPL1/ Male Duplicated Small unknown Mizuta et al., 2010

DPL2 genes proteins

Sc Male Copy number DUF1618 protein Shen et al., 2017

variation

HSA1a/ Male Two adjacent DUF1618 protein, Kubo et al., 2015

HSA1b genes unkown proteins

S7 Female Single gene TPR protein Yu et al; 2016

S1 Female/male Multiple genes TPR protein/unkown p Xie et al; 2017b

S1 locus-mediated hybrid sterilityin Asian-African rice hybrids

SS: Semi-Sterility

FF: Full Fertility

RP(S1-s) NIL(S1-g) F1(S1-s/S1-g)

SSFFFF

FF FF SS

Map-based cloning of S1

No. recombinant

Centromere(Chr.6)

*

1 2 3 4 5 6 7OsORF

1 5 10 15 17Exon

TCA TAA (stop codon)

*

2 000 0

Chromosomal position (kb)

2

OgTPR1

OsTP1

IF2 IF31 3 4 6 7OgORF a2 a3 a4 a5 a6a1

IF1 IF4 IF5

Trypsin-like peptidase RRS

Trypsin-like peptidase

(RRS, ribosome biogenesis regulatory protein domain)

S1(Xie et al., Mol Plant 2017)

Knocked out of OgTPR1 and OgORF7 did not affect male and female fertility

reference

Allele 1Allele 1

T0

(ogtpr1

)

D

CCATAG-CGATGCCTGAGCATG

CCATAGGCGATGCCTGAGCATGCCATAGGCGATGCCTGAGCATG

Aogtpr1 ogorf7B

reference

T0(ogorf7-1)

Allele 1Allele 1

TGGGGAACTTGGAC-TGGCGGTGGGGAACTTGGAC-TGGCGG

TGGGGAACTTGGACTTGGCGG

Knockout of OgTPR1 could rescue fertility in the interspecific hybrids

F1

NIL×RP-s

mF1

ogtpr1×RP-s

mF1

ogorf7×RP-s

PopulationGeno-type

No. plant

Pheno-type

χ2

(1:2:1)

g/gg/ss/s

FFFFFF

7015575

0.50mF2 of

(ogtpr1×RP-s)

mF2 of(ogorf7×RP-s)

g/gg/ss/s

FFSS

-

180150

472**

F2 of(NIL-g×RP-s)

g/gg/ss/s

FFSSFF

1194136

52694**

The S1-hybrid sterility involves at lest three linked genes

IF2 IF3

1 3 4 OgTPR1 OgORF7OgORF a2 a3 a4a1

IF1 IF4 IF5

* * * *

mF1-ogorfa2 mF1-ogorfa4 mF1-ogorfa5 mF1-ogorfa6

hypothetical

proteinhypothetical

protein

hypothetical

proteinAP2

TF

Typsin-like

proteinTypsin-like

protein

*Fertility-rescued

a5 a6

Mutant hybrids

Fertility-rescued

Sc-hybrid showed semi-male sterility and produced severe allele transmission ratio distortion (TDR)

FF: Full fertility;SS: Semi Sterility

• T65 (Sc-j/Sc-j): a japonica cultivar Taichung 65

• E5 (Sc-i/Sc-i): a near-isogenic line of T65 with Sc-i

Pollen grains carrying the Sc-j allele were selectively aborted

Map-based cloning revealed a large structural change and copy number variation (CNV) at Sc

Sc-i is a recombinant new gene with CNV, and the promoter regions between Sc-j and Sc-i differ completely

Sc locus90.5

0 BAC: OSJNBb0078P24

80.4

5

84.485.792.093.0100.7

2 0 24No. recombinant:(from 8689 F2 plants)

38.0114.0Marker (kb):

0

E14-E2RG227

Chr. 3

Marker

8.6 kb

Sc-j (Os03g0247300)

japonica cv: T65, Nip, Koshihikari,

Hitomebore, HEG4, A123

Sc-ib2Sc-is

28.4 kb

T1 T2

E5

Sc-ib1MH63

93-11

14.9 kb

Sc-iaSc-ib2 Sc-ib1Sc-is

28.2 kb28.4 kb

indica cv:

23.9 kb 23.9 kb

23.9 kb

Sc alleles encode diverged DUF1618 proteins

VHTFPFPVSGVVWVGRAAVLRHAGGGGGGGDGTTASYVIAELLRPFHGSLPDATLVMWLS 180

VHTFPFPVSGVVWVGTAAVLRHAGGGGGG-DGTTA-YVIAELLRPFHGSLPDATLVMWLS 177

PIISRFSMLISRKAIRVVKSVRVRCADKSLVLFYAGTGFPGFSSHGCHLIYDAIDGSLTA 120

PIVSRFSMLISRKAIRVVKSIHVECADKSLVLFYAGTGFPGFS-HGCHLIYDAIDGSLTA 119

MAPTWVLLDRFVKPTIFDEEESKGKGESTGAPVKYLPARLRQEVPAGMRDVKPYPEVADP 60

MAPAWVLLDRVVKPAVFDEEESKGKGESTGAPVKYLPARLRQEVPAGMRDVKPYPEVADP 60

MAPAWVLLDRVVKPAVFDEEESKGKGESTGAPVKYLPARLRQEVPAGMRDVKPYPEVADP 60

Sc-jSc-ia

Sc-ib1/ib2

PIVSRFSMLISRKAIRVVESIHVECADKSLVLFYAGTGFPGFS-HGCHLIYDAIDGSLTA 119

Sc-jSc-ia

Sc-ib1/ib2

VHTFPFPVSGVVWVGTAAVLRHAGGGGGG-DGTTA-YVIAELLRPFHGSLPDATLVMWLS 177

Sc-jSc-ia

Sc-ib1/ib2

NSPASTSGSNGQWVKEDVRLPGEVCTGTDPFTTDLVFSFGESCLCWADLFMGILFCDLAT 240

NSPASTSGSNGQWVKEDVRLPGEVCTGTDPFTTDLVFSFGESCLCWADLFMGILFCDLAT 237

NSPASTSGSNGQWVKEDVRLPGEVCTGTDPFTTDLVFSFGESCLCWADLFMGILFCDLAT 237

LRAPRFRFIPLPKACSFDPVGKYGRPHMPEFRSMGRVNGVIRLIDMEGFTNEYLAVDEVK 300

LRAPRFRFIPLPKACSFDPVGKYGRPHMPEFRSMGRVNGVIRLIDMEGFTNEYLAVDEVK 297

LRAPRFRFIPLPKACSFDPVGKYGRPHMPEFRSMGRVNGVIRLIDMEGFTNEYLAVDEVK 297

Sc-jSc-ia

Sc-ib1/ib2

Sc-j

Sc-iaSc-ib1/ib2

LTIWTLSADLSEWEKGPVCTVGDIWASEEFVAMGLPQLRPMCPVLSMVDEDVVCVVMTEV 360

LTIWTLSADLSEWEKGPVCTVGDIWASEEFVAMGLPQLRPMCPILSMVDEDVVCVVMTEV 357

LTIWTLSADLSEWEKGPVCTVGDIWASEEFVAMGLPQLRPMCPVLSMVDEDVVCVVMTEV 357

Sc-j

Sc-iaSc-ib1/ib2

EIEESDVTDFDDEGNKLKFKAQYVLDIDVRRKRVLSITQHHIESMGDLIPDLIACEFTAY 420

EIEESDVTDFDDEGNKLKFKAQYVLDIDVRRKRVLSITQHHIESMGDLIPDLIACEFTAY 417

EIEESDVTDFDDEGNKLKFKAQYVLDIDVRRKRVLSITQHHIESMGDLIPDLIACEFTAY 417

Sc-jSc-ia

Sc-ib1/ib2

SELSKGMQAMVEGNEGEESTKRMKVK 446

SELSKGMQAMVEGNEGEESTKRMKVK 443

LELSKGMQAMVEGNEGEESTKRMKVK 443

Sc-j

Sc-iaSc-ib1/ib2

DNA fiber-FISH verified the Sc-i CNV

indica cultivars have different copies (2 or 3) of Sc-i

Structures of Sc-j and the duplicated Sc-i paralogs

Sc-ia

Sc-ib1

Sc-j

Sc-ib2

Exon1(1380 bp)

Exon2(62 bp)

P6

P727-bp deletion

P1

P2

P5

P7

P7

P5

243-bp insertion

3x3-bp deletion

243-bp insertion

Promoter PSc-j

Promoter PSc-ia

PSc-ib

PSc-ib

A G G C C

A G G C C

G A A T T

G A A T T

P7

P6 P5

P5P6

P2

P2

P2

1363 bp

451 bp

ATG

Intron (1509 bp)

P3

P3

P3

P3

P8

P8

P8

P8

P4

P4

P4

P4

TGA

(RiceXPro,

http://ricexpro.dna.affrc.go.jp/)

Sc-j and Sc-i have different expression patterns and levels

OsActin 1 (25 cycles)

Sc

(28 cycles)

M

T65 E5a

kb

1.00.75

0.5

1.00.75

0.5

b

OsActin 1

Sc-j

Mean value = 2413 in 1.6-2.0 mm anthers

Mean value = 58620 in 1.6-2.0 mm anthers

Sc-j : Actin 1 = 0.04

Sc-j and Sc-i have different expression patterns and levels

x10-2

1

7

6

0Expr

essi

on (S

c-j/A

ctin

1)

5

Sc gene is a gametophytic factor for bicellular pollen development

e

0

0.5

0.25

x10-2

0.75

1.0

Rel

ativ

e S

c-j

expr

essi

on

**

Transgenic

lineT-DNA

site

Phenotype in T0

No.

plants (T1)Segregation in T1

(T+ : T-)

χ2

(3:1) (1:1)

Anti-1

Anti-2

Anti-3

Anti-4

Anti-5

1

1

1

1

1

SS

SS

SS

SS

SS

110

50

29

37

37

54:56

29:21

12:17

21:16

22:15

39.38***

7.71**

17.48***

6.57*

4.77*

0.04

0.32

0.86

0.68

1.32

Hemizygous anti-Sc-j

Higher Sc-i copy number produced more severe TRD in different japonica-indica crosses

Cross F1 genotype No. plants Segregation % jj plants χ2

E5/T65

ZS97B/Nip

Nip/HHZ

Nip/93-11

Dular/Nip

Nip/02428

F2 jj:ji:ii Exp 25% (1 : 2 : 1)

3xSc-i/Sc-j

3xSc-i/Sc-j

Sc-j/2xSc-i

Sc-j/2xSc-i

2xSc-i/Sc-j

Sc-j/2xSc-i

748

213

134

258

205

172

3:377:368

7:118:88

6:65:57

25:145:88

7:113:85

12: 95:65

356.3***

64.1***

40.7***

34.7***

61.5***

34.6***

0.4

3.3

4.7

9.7

4.4

7.0

RIL jj:ii Exp 50% (1 : 1)

106 (F10)

247 (F7)

1:105

52:195

0.01

21.1

102.0***

82.8***

GLA4/Nip

Nip/93-11

3xSc-i/Sc-j

Sc-j/2xSc-i

♀/♂

The Sc-j expression in the hybrid was largely suppressed

Early-bicellular anthers

Purified early-bicellular pollen

T65 F1

0.25

0.5

0.75

1.25

1.0

x10-2

Rel

ativ

e S

c-j

expr

essi

on

0

x10-2

0.5

1.0

1.5

2.5

2.0

T65 F1

0

***

(The expression levels were calculated as per Sc-j copy)

Deletion and mutation of one or two of the Sc-i copies by CRISPR/Cas9 editing

E5-d1Sc-iaSc-ib1Sc-is 28.4-kb deletion

ATTTCGCGGAAGGCGAT------AGTGGAATCCA

60 bp

ATTTCGCGGAAGGCGATCAGGGTTAGTGGAATCCA

E5-ed1Sc-ib1ed1

Sc-ib2ed1

E5-ed2Sc-ib1ed2

Sc-ib2ed2

P9c

P9d

GATTTCGCGGAAGGC-------------GAATCCA

AAGCCCTACCCAGA GGTAGTGGAATCCA--- ---

P9b

P9e

13 bp

ATTTCGCGGAAGGCGATCAGGGTAGTGGAATCCA

ATTTCGCGGAAGGCGATCAGGGTAGTGAAATCCG

PAM

Sc-ib1/ib2

Sc-iaE5

exon 1

P10

P9a

The edited Sc-i alleles are hybrid-compatible, overcoming male fertility in the hybrids

(1 copy deleted) (2 copies mutated) (2 copies mutated)

D E

0.25

0.5

0.75

1.25

1.0

x10-2

0

*

****

Exp

ress

ion

pe

r co

py

(Sc-j

/Act

in 1

)

0.1

0.05

0.025

0

0.075Sc-ia

Sc-ib1ed1

Sc-ib1ed2

Sc-ib2ed1

Sc-ib2ed2

Sc-ia/ib1/ib2

Exp

ress

ion

(S

c-i

/Act

in 1

)

The edited hybrids had normal male fertility and genotypic segregation

Indica-japonica hybrid male sterility at the Sc locus is caused by the duplications of the indica allele gene Sc-i with a gene-dosage effect

(Shen et al., Nat Commun 2017)

Sc-i acts as both the Sc-j-pollen killer and the self-protector

This molecular model well explains the classical “one-locus sporo-gametophytic interaction” model for hybrid sterility

Allelic suppression

SaF SaM

T860 (E4) AG(E4) TGA (T65)

TGA (E4)ATGATG TAGC860 (T65) AT(T65)

P0013G02 (163.4 kb)

A10-2 G02-148G02-45Centromere

~5.4 Mb

63

Recombinant Phenotype

E4

T65

G392 MSS

MSS

MFFA408

H70 MFF

MFFC513

MFFH70T65

MFFMFFG735MFFA408T65

H122

MSS

MSSC513H70

370 kb

69 79.6 (kb)78.576.374.6737269.8G02-

H71

MFFLS38

G02-84

(Chr. 1)

*

*

*

*

SaF SaM

T860 (E4) AG(E4) TGA (T65)

TGA (E4)ATGATG TAGC860 (T65) AT(T65)

P0013G02 (163.4 kb)

A10-2 G02-148G02-45Centromere

~5.4 Mb

63

Recombinant Phenotype

E4

T65

G392 MSS

MSS

MFFA408

H70 MFF

MFFC513

MFFH70T65

MFFMFFG735MFFA408T65

H122

MSS

MSSC513H70

370 kb

69 79.6 (kb)78.576.374.6737269.8G02-

H71

MFFLS38

G02-84

(Chr. 1)

*

*

*

*

The heterozygous SaM and at least one E4 (indica) SaF allele are required for the hybrid male sterility.

Male Semi-Sterility

Male Full Fertility

The Sa complex locus for japonica-indica hybrid male sterility (Long et al., PNAS 2008)

Knockout of SaF or SaM of the Sa locus could rescue fertility in japonica-indica hybrids

(Xie et al. JIPB 2017)

Knock out of SaF or SaM

did not affect male fertility

The mutant japonica-indicahybrids with mutant saf or samshowed normal male fertility

Rapid breeding of glutinous rice by editing Waxy

(Ma et al., MP 2015)

WT T0-1 T0-2 T0-3 T0-4

Knockout of the transporter genes (OsGSTU, OsMRP15, and OsAnP) in a purple-leaf rice destroyed the anthocyanin biosynthesis pathway

Summary

• The hybrid sterility loci often include multiple functionally-related genes

• The CRISPR/Cas9 toolkit is powerful for genome editing in plants

• The genome editing technology is very useful for study of gene function and genetic improvement, especially for breaking down of the reproductive barrier in hybrid breeding

ACKNOWLEDGMENTS

• Ma Xingliang, Xie Xianrong, Liu Weizhi, Shen Rongxing, other members of our lab

• MOST of China