fatima naim - exploring and improving genome editing in plants · exploring and improving genome...
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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