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 Xingyou.gu@sdstate.edu MW American Society of Plant Biologists Meeting March 20th 2016 Brookings, SD

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

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

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

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

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

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

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

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)

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

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%

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

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

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

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

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

THANK YOU!!