Genome editing in plants using small Cas9 and FnCpf1 - PowerPoint Presentation

Slide1

Genome editing in plants using small Cas9 and FnCpf1

1. Institute of Agrobiological Sciences, NARO, Japan. 2. Graduate School of Nanobioscience, Yokohama City Univ., Japan. 3. Kihara Institute for Biological Research, Yokohama City Univ., Japan.

Akira Endo1, Hidetaka Kaya1 and Seiichi Toki1,2,3

Slide2

Part 1: Targeted mutagenesis using SaCas9 in tobacco

Part 2: Targeted mutagenesis using Split-SaCas9 in N. benthamianaPart 3: Targeted mutagenesis using FnCpf1 in tobacco and rice

Today’s topics

Slide3

5’

sgRNA

(artificial)

5’

5’

3’

3’

5’

3’

NGG

PAM

tracrRNA

crRNA

5’

3’

Target sequence

20

nt

guide

sequence

NGG

PAM

3’

Classification: Class II type II

Number of amino acid residues

：

approx. 1000~1400

DNA end after cleavage

：

Blunt end

crRNA: 42

nt

tracrRNA

: 75

nt

Protospacer

Adjacent Motif (PAM)

：

NGG

(

Cas9 from

Streptcoccus

pyrogenes

)

Single guide RNA (

sgRNA

: approx. 100

nt

)

Cas9 is a RNA-directed endonuclease

Slide4

sgRNA

Cas9 protein

sgRNA

and Cas9 protein complex

Targeted mutagenesis using Cas9 in model plant

Bind to genome DNA

Cleave the target sequence

T-DNA delivery into plant genome via

Agrobacterium

Cleaved DNA is mainly repaired by non-homologous end

joining (NHEJ),

error-porn DNA repair pathway.

Translocation from cytosol to nucleus

Slide5

Part 1: Targeted mutagenesis using SaCas9 in tobacco

Part 2: Targeted mutagenesis using Split-SaCas9 in N. benthamianaPart 3: Targeted mutagenesis using FnCpf1 in tobacco and rice

Today’s topics

Slide6

SpCas9

Streptococcus pyogenes SaCas9Staphylococcus aureus

4.1 kb3.2 kb

gene size

PAM

NGG

NNGRR(T)

target sequence

20

nt

24 – 21

nt

R: A or G

S

a

Cas9 is smaller than SpCas9

(Kaya

et al

, Scientific Reports, 2016)

Slide7

target

　geneCas9No. of examined plants

No. of plants with mutationsMutations rate (%)

S

a

Cas9

45

34

75.6

NtPDS

SpCas9

44

35

79.5

S

a

Cas9

43

28

65.1

NtFT4

SpCas9

22

1359.1

Nicotiana tabacum

wild type

pds

mutant

1 cm

The mutation rate of S

a

Ca9 is identical to that of SpCas9

(Kaya

et al

, Scientific Reports, 2016)

PDS: Phytoene Desaturase

Slide8

No. of

target sequence PAM(Sa) in/del clonesCCAAGCCCAAGCAACCCTAACCTGAGGGAGTATCT WT CCAAGCCCAAGCAACCCTAACC-GAGGGAGTATCT -1

×2CCAAGCCCAAGCAACCCTAA--TGAGGGAGTATCT -2 ×4CCAAGCCCAAGCAAC-------TGAGGGAGTATCT -7

×

1

CCAAGCCCAAGCAACCCTAACC--------TATCT -8

×

1

CCAAGCCCAAGCAACCCT----------AGTATCT -10 ×

1CCAAGCCCAAGCAACCCTAACC

t

TGAGGGAGTATCT +1

×2

CCAAGCCCAAGCAACCCTAACC

cTGAGGGAGTATCT

+1

×2

(total: 13)

CCAAGCCCAAGCAACCCTAACC

t

TGAGGGAGTATCT +1

×18

(total: 18)#18:

#24:#17:#5:SaCas9SpCas9 No. of target sequence PAM(Sp) in/del clonesCCAAGCCCAAGCAACCCTAACCTGAGGGAGTATCT WTCCAAGCCCAAGCAACCCTAACCT

t

GAGGGAGTATCT +1

×

13 (total: 13)

CCAAGCCCAAGCAACCCTAAC----------ATCT -10

×13

CCAAGCCCAAGCAACCCTAACCcTGAGGGAGTATCT

+1 ×

1 (total: 14)

Target gene: NtFT4

gene

Patterns of mutation induced by

Sa

Ca9 or SpCas9

(Kaya et al

, Scientific Reports, 2016)

Slide9

PAM mismatch

NtFT4: GCCCAAGCAACCCTAACCTGA GGGAGT

NtFT1: CCCCAAGCAACCCAAACCTGA

GG

GAGT

2

bp

NtFT2: C

CCC

GAGCAACCC

AAA

TCTGA GG

GAGT

4 bp

S

aCas9

NtFT2

(off target)

NtFT1 (off target)

SpCas9

0 / 32

0 / 9

NtFT4 (on target)19 / 326 / 90 / 321 / 9 PAM mismatchNtFT4: CCCAAGCAACCCTAACCTGA GGG

NtFT1

:

CCCAAGCAACCCA

AACCTGA G

GG 1 bp

NtFT2

: CCCGAGCAACCC

AAAT

CTGA G

GG 3

bp

S

aCas9

SpCas9

Differences of Off target mutation

ratio between S

aCas9 and SpCas9

(Kaya

et al

, Scientific Reports, 2016)

Slide10

Specificity for target sequence

SaCas9>SpCas9Targeted mutagenesis efficiency

SaCas9=

SpCas9

Summary of part 1

Slide11

Part 1: Targeted mutagenesis using SaCas9 in tobacco

Part 2: Targeted mutagenesis using Split-SaCas9 in N. benthamianaPart 3: Targeted mutagenesis using FnCpf1 in tobacco and rice

Today’s topics

Slide12

Agrobacterium

Retorovirus

Nucleus

T-DNA

T-DNA

T-DNA

Cas9

1

2

3

4

Virus system does not necessary to integrate

DNA fragment

encoding Cas9 into plant genome

Nucleus

Cas9

1

2

3

Limited size of foreign DNA can

be mount

on virus vector (approx. 2

kbp

)

Slide13

SaCas9-430N

SaCas9-431C

SaCas9-

739N

SaCas9-

740C

430

a.a

.

739

a.a

.

314

a.a

.

Application of Split-SaCas9 to plant genome engineering

(Kaya

et al

, Plant Cell & Physiol., 2016)

(

Nishimasu

et al

., Cell, 2015)Domain structure of SaCas9

623

a.a

.

Slide14

N. benthamiana

35S

-pro::split SaCas9_430N

(or

739N

)

35S

-pro::split SaCas9_431C

(or

740C

)

AtU6

-pro::

sgPDS1

–

+

+

+

+

+

+

+

+

+

+

sgPDS1

mock

empty

vector

SaCas9

430N/ 431C

739N/ 740C

Bst

NI

undigested

band

Marker

Efficiency of Targeted

Mutagenesis (%)

SaCas9

split SaCas9

430N/ 431C

split SaCas9

739N/ 740C

split SaCas9

Evaluation of genome editing activity

of split-SaCas9 by

Agrobacterium

infiltration

(Kaya

et al

, Plant Cell & Physiol., 2016)

(indicating mutation)

Slide15

N. benthamiana

ToMV

vector

Agrobacterium

Extraction of

genomic DNA

and

CAPS

analysis

Trial to mount one of split-SaCas9 on the virus

vector

ToMV

: Tomato Mosaic Virus

4 days

7

days

Slide16

SaCas9

(Agro)SaCas9 (ToMV)430N (

Agro) /431C (ToMV)430N (ToMV) /

431C (Agro

)

739N

(

Agro) /740C

(ToMV)

739N (ToMV

) /740C (

Agro)

undigested

band

+

+

+

+

+

+

+

Bst

NI

split SaCas9Marker12575Evaluation of genome editing activity of split-SaCas9 by

Agrobacterium

infiltration followed by virus infection

(Kaya

et al, Plant Cell & Physiol., 2016)

(indicating mutation)

Slide17

Split-SaCas9 represent the genome

edting activity in N. benthamianaSaCas9 split-SaCas9

_739N/_740Csplit-SaCas9_

430N/_431C

=

>

Genome editing activity of virus-derived split-SaCas9_739N/_740C was confirmed.

Summary of part 2

Slide18

Part 1: Targeted mutagenesis using SaCas9 in tobacco

Part 2: Targeted mutagenesis using Split-SaCas9 in N. benthamianaPart 3: Targeted mutagenesis using FnCpf1 in tobacco and rice

Today’s topics

Slide19

5’

5’

3’

3’

5’

3’

crRNA

TTN

PAM

5’

3’

NGG

PAM

tracrRNA

Cpf1 Cas9

Classification: Class II type V Class II type II

Number of amino acid residues

：

approx. 1300 approx. 1000~1400

DNA end after cleavage

：

Sticky end

( 5’ overhang) Blunt end

crRNA:

43

nt

42

nt

tracrRNA

: --- 75

nt

PAM

：

TTN

(FnCpf1) NGG (SpCas9)

TTTN (As, LbCpf1) NNGRRT(SaCas9)

FnCpf1

SpCas9

crRNA

5’

3’

Target sequence

guide sequence

New RNA-directed endonuclease, Cpf1

Cpf1 from

Francisella

novicida

Slide20

Possible application of Cpf1

AT-rich regionGC-rich region

Cpf1

Cas9

PAM preference

Slide21

ATGTCTATAAATAT

AAGAGACCCTCTTATAGTAAGCAGAGTTGTTGGAGACGTTCTTGAT

CCGTTTAATAGATCAATCACTCTAAAGGTTACTTATGGCCAAA

GAGAGGTGACT

AA

T

GG

CTTGGATCTAAGGCC

TTCTCAGGTTCAAAA

CAAGCC

AAGAGTT

GAGATTGGT

GGAGAA

GACCTCA

GGAACTTCTATAC

TTTGGTT

ATGGTGG

ATCCAGATG

TTCCAA

GTCCTAGCAA

CCCTCACC

TCCGAGAA

TATCTCCATTGGTTGGTGACTGATAT

CCCTGCTACAACTGGAACAACCTTTGGCAATGAGATTGTGTGTTACGAAAATCCAAGTCCCACTGCAGGAATTCATCGTGTCGTGTTTATATTGTTTCGACAGCTTGGCAGGCAAACAGTGTATGCACCAGGGTGGCGCCAG

AAC

TTCAA

CACTCGCGAGTTTGCTGAGATCTAC

AATCTCGGCCTT

CCCGTGGCCGCAGTTTTCTAC

AATTGTCAGAGGGAGAGTGG

CTGCGGAGGAA

GAAGACTTTAG

528 bp region from Arabidopsis genome

SpCas9 (

NGG): 58 sites

FnCpf1 (

NTT): 81 sites

PAM of SpCas9 and FnCpf1 evenly cover the target region

Slide22

Precise genome editing in vivo ligation with 5’ sticky end generated by Cpf1

Knock-inReplacementPossible application of Cpf1

Slide23

FnCpf1-mediated targeted mutagenesis in

tobacco and riceBinary vector

Experimental schemeTobacco

Tobacco

Rice

Rice

Slide24

WT(S) ACTTGCT

TTCTCATCCAGTCCTTAACACTTAAACCGTCTTGAGCWT(T) ACTTGCTTTCTCATCCAGTCCTTAACACTTAAACCGTCTTGAGC

#12(S) ACTTGCTTTCTCATCCAGTCCTTAACACTTAA-CCGTCTTGAGC -1 x2 (65.2) ACTTGCTTTCTCATCCAGTCCTTAAC--TTAAACCGTCTTGAGC -2 x13/23 (T) ACTTGCTTTCTCATCCAGTCCT--------AAACCGTCTTGAGC -8 x3/24 (12.5)#14(S) ACTTGCTTTCTCATCCAGTCCTTA--------ACCGTCTTGAGC -8 x3/24 (12.5)

(T) ACTTGCTTTCTCATCCAGTCCTTAAC------ACCGTCTTGAGC -6 x1 (14.2)

ACTTGCTTTCTCATCCAGTCCTT--------AACCGTCTTGAGC -8 x2/21

Target sequence

PAM

Mut

. Freq.(%)

Indel

1 2 3

4 5

6

7

8 9

10

11

12

13

14

15 16 17

18 W

W W W W W

NtPDS

(S)

NtPDS (

T)

Detection of mutation using HMA

Heteroduplex

Homoduplex

Heteroduplex

Homoduplex

Patterns of mutations

FnCpf1-mediated targeted mutagenesis in tobacco

(Endo

et al

, Scientific Reports, 2016)

(indicating mutation)

S

and

T

indicate

N

.

sylvestris

and N.

tomentosiformis

#of transgenic line

(

PCR product without mutation

)

Slide25

FnCpf1-mediated targeted mutagenesis in rice

OsDL

-2

WT GTG

TTA

GGGACCTTGCACTGACTGCAGGAG

GAACCAGCCG

#2 GTGTTAGGGACCTTGCACTGACTG---GAGGAACCAGCCG -3 x1 (75) GTGTTAGGGACCTTGCACTGAC---AGGAGGAACCAGCCG -3 x2

GTGTTAGGGACCTTGCACTGACTG-----GGAACCAGCCG -5 x2 GTGTTAGGGACCTTGCACTGA-------AGGAACCAGCCG -7 x2 GTGTTAGGGACCTTGCACTGACTG-------AACCAGCCG -7 x1

GTGTTAGGGACCTTGCACTGA--------GGAACCAGCCG -8 x1

GTGTTAGGGACCTTGCAC---C-----GACGAACCAGCCG -8 x1 GTGTTAGGGACCTTGCACTGA--------GGAACCAGCCG -8 x2

GTGTTAGGGACCTTG----G--TG--TTAGGAACCAGCCG -8 x1 GTGTTAGGGACCTTGCACTG----------------GCCG -16 x1

GTGTTAGGGACCTTGCA-------------------GCCG -19 x1

GTGTTAGGGA-------------------GGAACCAGCCG -19 x1 GTGTTAGGGACC-----CTG-C--------//---AATGA -43 x1

GTGTTAGGGACCTTG----G--TG--TT

AGGAACCAGCCG -8m2 x1/24

Target sequence

PAM

Mut

. Freq.(%)

Indel

(Endo

et al

, Scientific Reports, 2016)DL: Drooping Leaf

Slide26

Summary of part 3

FnCpf1 could be applicable to targeted mutagenesis in rice and tobacco.Most of FnCpf1-induced mutations were deletion.

Slide27

FnCpf1

SpCas9

Virus A vector

Virus B vector

T-DNA integration free genome editing

Target most of genomic region

Precise genome editing

Knock-in

Replacement

Various nucleases broaden the possibility of genome editing in plant

SaCas9

Slide28

Members of Plant

Genome Engineering Unit Head of Unit : Dr. Seiichi Toki HidetakaSeiichiMasafumi

Acknowledgement

Slide29

Target gene

crRNATarget gene

PAMTarget sequence Mutation frequency of

calli (%)Exp. 1 Exp. 2

OsNCED1

OsNCED

-t1

OsNCED1

(

On)

TTC

CCCAAGGCCATTGGGGAGCTCCAT

21.4

23.3

OsNCED2

(Off)

TTC

CCCAAGGCCAT

C

GGCGAGCTCCAT

0

6.25

OsNCED3 (Off)TTCCCCAAGGCCATCGGCGAGCTCCAC 00OsAAO1OsAAO-t1OsAAO1 (

On)

TTG

GCAATGCTGTGTCATATGTTAATT

38.8

50

OsAAO2

(

On)TTG

GCAATGCTGTGTCATATGTTAATT

24.1

36.6

OsAAO3

(Off)

TTG

GCAATGCTGT

T

TCATATGTTAATT

0

0

OsAAO4

(Off)

TTG

GCAATGCTGT

TTCATATGTTAATT

<5

<5

OsAAO5

(Off)

TTGGCAATGCTGTCTCATATGT

GAATT 00Off-target mutation analysis in rice

(Endo et al, Scientific Reports, 2016)