Exploring and improving genome editing in plants

Fatima Naim

Kylie Shand

Ben Dugdale

Peter Waterhouse

@The Centre for Tropical Crops and Biocommodities

CRISPR-Cas9 editing tool

RuvC domain

HNH domain

Efficient in higher plants:

Effective in gene knockout resulting from

unpredictable indels

• Very inefficient in higher plants

• Desired for precise genome editing including:

• Gene knock in

• Restore dead genes

CRISPR-Cas9 plasmid design

2X35SSpCas9 AtU3 tRNA-gRNA cloning site enTCUP PPT

RB LB

Xie et al. (2015)

2X35SSpCas9 AtU3 PPT

RB LB

tRNA tRNAgRNA 1 2 TTT gRNA enTCUP

2X35SSpCas9 AtU3 PPT

RB LB

(a) pCas9-GFP

tRNA tRNAgRNA

Loss of 324 bp

1 2

CTTGTCACTACTTTCTCTTATGG5’ CGCCACAACATCGAAGACGGCGG 3’

(b) mGFP5-ER targets in 16c

(c) PCR (d) Sanger sequencing trace of pGEM clones

Original size of GFP, 736 bp

Excised fragment, 392 bp

- + + pCas9-GFP

+ + + p19

**

PAM PAM

TTT gRNA enTCUP

Editing of mGFP5-ER in 16c – Transient leaf assay

- + - - - - pCas9 (0.2)

- - + - - - pCas9 (0.5)

- - - + - - pCas9 (1.0)

- - - - + - pCas9 (1.5)

- - - - - + pCas9 (2.0)

Optimisation of agro concentration used in transient assay

OD600 nm

** ** *

Original size of GFP, 736 bp

Excised fragment, 392 bp p35S:GFP (0.2 OD600 nm)

4 dpi

Change of cell cycle with the help of viral bits

• Geminiviruses are a large family of plant viruses

• Single-stranded, circular DNA genomes (∼2.5–3.0 kb)

• Upon infection, they produce numerous replicons through rolling-circle replication

• Rep/RepA interfere with cellular components for the cell to transition from G1 to S-phase

Change of cell cycle with the help of viral bits

+ Rep/RepA (0.2)

+ + pCas9

+ + p19

Original size

Excised fragment3 dpi

Parameters for efficient editing

• Plant age: 4 weeks

• Leaf size: medium

• Suppressor: p19

• OD600 nm: 0.5–1

• Harvest tissue: 4–5 dpi

• Cell cycle change wasn’t successful with TYDV Rep/RepA

gRNAs targeting NbFAD2.1

pCas9-NbFAD2.1

pCas9-

NbFAD2

Targets PCR fragment Lost fragment PCR fragment Spacer and PAM (5’ – 3’)

pCas9-AB A – B 351 bp 170 bp 181 bp A: GAGGTTGGTACCTTTTC|AAG-AGG

pCas9-DE D – E 483 bp 242 bp 241 bp B:GGTAGCAATATGGGGAC|GGG-AGG

pCas9-BC B – C 533 bp 201 bp 332 bp C: GTGAGCCAGTGTTGGAG|TGG-TGG

pCas9-CD C – D 665 bp 201 bp 464 bp D: GCATGTCACTATGACCC|TTA-CGG

pCas9-AC A – C 533 bp 371 bp 162 bp E: GGATTGGCTAAGGGGAG|CTT-TGG

pCas9-AD A – D 995 bp 572 bp 423 bp

pCas9-AE A – E 995 bp 814 bp 181 bp

(b) PCR

* ** *

* ** *

* *

* *

* *

(a) gRNAs against NbFAD2

gRNAs targeting NbFAD2.1

Editing of NbFAD2.1 – fatty acid analysis

0

2

4

6

8

10

12

% F

AM

E o

f ole

ic a

cid

+ pCas9-AB

+ pCas9-DE

+ pCas9-BC

+ pCas9-CD

+ pCas9-AC

+ pCas9-AD

+ pCas9-AE

+ hp-NbFAD2-300 short

+ hp-NbFAD2-600 long

+ V2 + AtDGAT1

n = 4

Cutting and joining is very efficient

Online sgRNA design tools used to target NLUC

Target target site Broad Ins WU-CRISPR ZiFiT Benchling CCTop Benchling CCTop Dimitri Perrin

1 GACCAAGTCCTTGAACAGGG no order 6 10 5 50 22 26 5 - accepted

2 CAGTTTGTTTCAGAATCTCG no order 8 12 17 38 49 29 9 – rejected

3 TGTCCGTAACTCCGATCCAA 2 15 13 21 16 27 45 No ranking

4 CACCGCTCAGGACAATCCTT no order Rejected 67 69 25 19 18 15 – rejected

5 ATTGTCCTGAGCGGTGAAAA no order Rejected 15 66 47 21 35 11 – rejected

6 TAAAGTGATGATCATCCACA 5 1 60 1 52 58 63 No ranking

7 ATGGCACACTGGTAATCGAC no order Rejected 28 37 6 8 23 2 – accepted

8 TGGCACACTGGTAATCGACG 1 17 29 3 1 5 10 No ranking

9 AACACGGCGATGCCTTCATA no order Rejected 56 59 7 30 30 3 – accepted

10 GATGGTTACTCGGAACAGCA no order 2 48 7 29 13 13 1 – accepted

Reference

genomeDefault Default Default

Homo38

based on

specificity score

and not

efficiency score

homo38

Niben101 based

on specificity

score and not

efficiency score

Niben101benthgenome.qut.edu

.au

RB LBp35SpSSU NanoLucGFP

+

2X35SSpCas9 AtU3 PPT

RB LB

tRNA tRNAgRNA 1 2 TTT gRNA enTCUP

gRNAs targeting NLUC

NLUC reading for top ranking and rejected gRNA

10

5

10

10 10

5

55

66

6

6

33

33

88

8

9

8

7

1

2

4

Control97

1

2

4

Control

Control

42

19

7Control7

9

1

2

4

0.00E+00

1.00E+08

2.00E+08

3.00E+08

4.00E+08

5.00E+08

6.00E+08

7.00E+08

8.00E+08

Control 1 2 3 4 5 6 7 8 9 10

Comparison of various gRNAs against NLUC, 3 leaf analysis

top rankingrejected

Comparison of 2X human codon optimised Cas9s

2X35SSpCas9 (A) AtU3 NPTII

RB LB

tRNA tRNAgRNA 1 2 TTT gRNA enTCUP

NLS

AtU3 HYG

RB LB

tRNAtRNA gRNA 12TTT gRNA 35S

NLS

2X35SSpCas9 (B)

NLS

pCas9(A)

Control pCas9(B)

+ + + + + + + p19

+ + + - - - - pCas9(A)

- - - + + + - pCas9(B)

Dropout of 293 bp

Dropout of 293 bp

5’ 3’

NbFAD2 targets in N.benthamiana:

CCACTCTGCTCTGATGGTGCCCTCCTCCTTTCACAGTTGGTGATATPAM PAM

Comparison of editing constructs in stable transformations

SpCas9 (A) pU3

RB LB

tRNAtRNA gRNA 12TTT gRNA enTCUPp2X35S(i) pFN092

SpCas9 (B) pU3 HPTIIRB LB

tRNAtRNA gRNA 12TTT gRNA p35Sp2X35S(ii) pFN094

(iii) pFN142

(iv) pFN121

RB LBSpCas9 (B) pU3 NPTIItRNAtRNA gRNA 12TTT gRNA pNOSp2X35S

Hsp1.8 + CaMV 35S poly A terminator NOS terminator

RB LBSpCas9 (B) pU3 NPTIItRNAtRNA gRNA 12TTT gRNA pNOSp2X35S

Hsp1.8 + CaMV 35S poly A terminator NOS terminator

enTCUPV2

NOS terminator

NPTII

pFN092 pFN094 pFN142 pFN121

Target 1 0 17/49 (35%) 29/52 (56%) 17/51 (33%)

Target 2 0 22/49 (45%) 34/52 (65%) 24/51 (47%)

Dropout 0 5/49 (10%) 10/52 (19%) 6/51 (12%)

# lines edited 0/50 30/49 (61%) 44/52 (85%) 33/51 (65%)

Total edited events 0 44/49 73/52 47/51

Summary of edited T0 lines

Screening T0 for dropouts in NbFAD2.1 gene

Dropout of 293 bp

pFN142 lines showed the highest number of DNA dropouts (19%)

RB LBSpCas9 (B) pU3 NPTIItRNAtRNA gRNA 12TTT gRNA NOSp2X35S

Hsp1.8 + CaMV 35S poly A terminator NOS terminator

Dropout of 293 bp with extra nibbling

pFN142-21 homozygous biallelic editing at two target sites

pFN142-25 dropout with extra nibbling

pFN094-12 homozygous biallelic editing at one target site

Various DNA repair patterns

Species Gene Target 1 Target 2 Dropout Distance between gRNA1 and gRNA2

A.thaliana AtCHS 0/10 0/10 0 328 bp

A.thaliana mir166a 0/33 12/33 0 478 bp

N.Tabacum (NN) NtRDR1 0/11 6/11 0 450 bp

N.tabacum (LYND) NtRDR1 0/4 2/4 0 450 bp

N.benthamiana NbFAD2.1 (1) 0/2 0/2 0 170 bp

N.benthamiana NbFAD2.1 (2) 1/4 4/4 0 242 bp

N.Benthamiana 16c mGFP5-ER 0/6 1/6 0 324 bp

Editing in T0 A.thaliana, N.tabacum and N.benthamiana

Polyploidy and gene editing

Locus 1

Locus 1 Homeologous Locus 2

Polyploidy and gene editing

Quadallelic editing of RDR1 in Nicotiana tabacum

Locus 1 Homeologous Locus 2

2X35SSpCas9 AtU3 PPT

RB LB

tRNA tRNAgRNA 1 2 TTT gRNA enTCUP

Locus 1 Homeologous Locus 2

Insertion of “A”

Line

13-5

9-10

9-5

13-9

7-10

2 copies of RDR1

Quadallelic editing of RDR1 in Nicotiana tabacum

Line

9-7

9-3

9-9

9-1

9-6

9-2

9-4

9-8

Indels

Editing of RDR1 in N.tabacum

Locus 1 (AA genome) Locus 2 (A1 genome)

Gene editing in banana

pFN106 pFN108

Target 1 4/7 (57%) 0/16 (0%)

Target 2 7/7 (100%) 16/16 (100%)

Dropout 0 (0%) 0/16 (0%)

# lines edited 7/7 (100%) 16/16 (100%)

SpCas9 (B) pU3 HPTIIRB LB

tRNAtRNA gRNA 12TTT gRNA p35SpUbi(ii) pFN106

SpCas9 (B) pU3 HPTIIRB LB

tRNAtRNA gRNA 12TTT gRNA p35Sp2X35S(ii) pFN108

Editing MaPDS

Editing MaPDSLocus 1 (AA genome) Locus 2 (A1 genome)

0

10

20

30

40

50

60

70

80

90

100

pUbi-Cas9 p35S-Cas9

Albino

Pale green

Green

Summary

• Design gRNAs and look for off targets

• Currently working on generating gRNAs against all genes in N.benthamiana

• Incorporate rules described by WU-CRISPR

• Increase chance of gene disruption by using 2X gRNAs

• Cas9 with 2X NLS in a backbone with no repeated promoters and terminators works best

Thank you!

Contact:

Fatima.Naim@qut.edu.au

Peter.Waterhouse@qut.edu.au