1. Mutagenesis of Genes Associated with Seed Dormancy in Rice Using Two CRISPR/Cas9 Multiplex Systems
Alexander Kena, Heng Ye, Jiuhuan Feng, Xingyou Gu
Seed Molecular Biology Lab.
Department of Plant Science
MW American Society of Plant Biologists Meeting
March 20th 2016
Brookings, SD

2. Concepts of seed dormancy
Seed dormancy (SD) refers to a temporary hold on germination of an intact viable seed under favorable conditions
SD has biological or agricultural significance:
Pre-harvest sprouting (weak or no SD)
Weed persistence (strong SD)
Understanding the genetics and molecular mechanisms of SD very crucial in solving real agricultural problems

3. Understanding the genetics of SD
using rice as a model species to identify SD QTLs X
cultivated rice (weak SD)
weedy rice (strong SD)
Germination distribution of BC1 EM93//EM93/SS18 population after 11 days of after-ripening
Number of lines
Percentage germination
Gu et al. (2004): Genetics 166, 1503–1516

4. SD QTLs identified in our lab
10 SD QTLs identified; 5 map-based cloned
qSD1-1
qSD7-1
qSD10
qSD8
qSD4
qSD6
qSD7-2
qSD12
qSD1-2
qSD3
SD QTL identified in rice
Linkage map developed based on BC1 EM93//EM93/SS18 population
Gu et al. (2004): Genetics 166, 1503–1516

5. Do these cloned genes really control SD?
Loss of function analysis through mutagenesis
Chr 4
Chr 7
Chr 12
qBh-4 collocates with qSD4
LOC_Os04g38660
qSD7-1/Rc
LOC_Os07g11020
qSD7-2
qSD12a, b and C
Objective
To disable these genes via a CRISPR/Cas9 system to generate mutants and observe phenotype of the derived mutants

6. Site-directed mutagenesis via CRISPR/Cas9
CRISPR/Cas9 system is an efficient genome editing tool
Clustered Regularly Interspaced Short Palindromic Repeats
Gene model for SD7-1/Rc: LOC_Os07g11020 (Gu et al. 2011)
Cas9 nuclease 5ˊ 3ˊ
gRNA
||||||||||||||||| |||
UGGCAGCUCUGCCCCCA CCA
DNA
Target sequence
PAM
(NGG)
5ˊ--TCCTCTGGCAGCTCTGCCCCCA CCAAGGGTACCT--3ˊ
|||||||||||||||||||||| ||||||||||||
3ˊ--AGGAGACCGTCGAGACGGGGGT GGTTCCCATGGA--5ˊ
Gu et al. (2011): Genetics 189, 1515–1524

7. CRISPR/Cas9 multiplexing
Designed two CRISPR/Cas9 multiplex constructs
Cas9 nuclease 5ˊ 3ˊ
gRNA
||||||||||||||||| |||
UGGCAGCUCUGCCCCCA CCA
Cas9 nuclease 5ˊ 3ˊ
gRNA
||||||||||||||||| |||
ACUCCUUCCCUCCCGGC AUC
DNA
Target sequence
PAM
(NGG)
Target sequence
PAM
(NGG)
5ˊ--TCTGGCAGCTCTGCCCCCA CCAAGGGTAC-----CCTACTCCTTCCCTCCCGGC ATCGGGTA--3ˊ
||||||||||||||||||| |||||||||| |||||||||||||||||||| ||||||||
3ˊ--AGACCGTCGAGACGGGGGT GGTTCCCATG-----GGATGAGGAAGGGAGGGCCG TAGCCCAT--5ˊ  
U6a
gRNA
U6b
U6c
SD7-1
Bh4
SD7-2
SD12c
SD12a
SD12b
6 target sites: one cleavage site per target gene
U6a
U6b
U6c U3
SD7-1_1
SD12a_1
Bh4_1
SD7-2_1
SD12b_1
SD12c_2
SD7-1_2
SD12a_2
Bh4_2
SD12c_1
SD7-2_2
SD12b_2
12 target sites: two cleavage sites per target gene
Ma et al. (2015b): Mol. Plant 8, 1274–1284

8. Pipeline for generating CRISPR/Cas9 mutants
Design of gRNA constructs
Assembly of Cas9 & gRNA
Delivery to plant
Agrobacterium-mediated plant transformation of cv. Nipponbare
Regenerate T0 plants for mutants screening
23 independent transgenic events for the two CRISPR multiplex systems
Ma et al. (2015b): Mol. Plant 8, 1274–1284

9. Decoding sequences for mutant alleles
DSDecode: web-based software (dsdecode.scgene.com/home)
Chromatogram from a mutant plant
Overlapping peaks
Degenerate sequence
GC|TC|GT|G|G
Bh4_1 cleavage site
Chromatogram from Nipponbare (Reference sequence)

10. Summary of mutant types
Mutant types differed with respect to target genes for 1-Target-site multiplex system
N=32

11. Summary of mutation types
InDel types constituted 95% of all mutations for 1-Target-site multiplex system
Deletions
Insertions
Gene -1 -2 -3 -4
-5~20
>20 +2
+A/T
+G/C
subs
Total
Bh4 11 5 4 1 26
SD7-1 2 9 3 12 40
SD7-2 13 7 49
SD12a 8 41
SD12c 18 42 32 28 21 16 44 19 10
198
Freq. (%)
62%
33% 5%

12. 1- vs 2-Target-site multiplex systems
Mutation rate differed with respect to target genes for 1-Target-site multiplex system
N=32
p = 0.009

13. 1- vs 2-Target-site multiplex systems
Targeting efficiency for 5 target genes was higher in the 2-Target-Site CRISPR/Cas9 multiplex system
P = 0.09
N=32

14. Future directions of on-going work
Observe effects of CRISPR/Cas9 induced mutations on functional SD genes under weedy rice genetic background
SS18-2
(weedy rice) × F1 F2
CRISPR/Cas9 T0 plant
This technique can be used to develop a strategy to mitigate flow of transgenes from GMOs to conspecific weedy relatives

15. Transgene-flow mitigation strategy
Using SD as a mitigating factor to develop a transgene-flow mitigation strategy
Goal is to neutralize any fitness advantage imparted by transgene in hybrids by silencing key SD genes

16. Acknowledgements Supervisor & PI Dr. Xingyou Gu Lab members
Dr. Jiuhuan Feng
Mr. Wirat P.
Mr. Luai Muhammad
Mrs. Ismail Fatma
Mr. Ahmed Charif
Dr. Heng Ye
University of Missouri

17. THANK YOU!!