F-LDHU GGATCCAATGTTATGTTGCGAATATATTTTAAATAA R-LDHU GGATCCAAGATCCTCCAAAATTTATATTACCA F-LDHD CTGCAGGACTTATTGACTAAGCTCGGG R-LDHD CTGCAGTTTCATGGCACCAAGTGC IF-stCAT GGACTTCATTTACTGGGTTT OR-ldh ATATTGTGCAATGAAACCAGC F1-v4sfgsgR TGAGTATAACCAGAACATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAG CAAGTTAAAATAAGG F2-v4sfgsgR AAGTTAATAGGTGTTTTTAGCCTGAAGTGTTATAATGAGTATAACCAGAACATCT AATTCAACAA F3-v4sfgsgR CCGGCGCCGAGAACAGTTTAAAGTTAATAGGTGTTTTTAGCCTGAAGT R-v4sfgsgR GGCGCCAGTTCACCGACAAACAACAGAT F1-v4folEsgR CAGAAATATTTGACGCCCTGCACGAGTTTTAGAGCTAGAAATAGCAAGTTAAA ATAAG F2-v4folEsgR AGTTAATAGGTGTTTTTAGCCTGAAGTGTTATAATGAGTATAACCAGAAATATTT GACGCCCTGC F3-v4folEsgR AATCTAGAGAGAACAGTTTAAAGTTAATAGGTGTTTTTAGCCTGAAGT F1-v4ribsgR AATGAGTATAACCAGAAGGCTGTTTGAAATTTTGAATGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAG F2-v4ribsgR GAGAACAGTTTAAAGTTAATAGGTGTTTTTAGCCTGAAGTGTTATAATGAGTATA ACCAGAAGGCTGTTTG F1-eSD2dCas9 ATCTTAATCATGGAAGGGAGGGTTTTTAATGGATAAGAAATACTCAATAGGCTT AGCTA F2-eSD2dCas9 GGCTCGAGATGAAAGCAATTTTCGTACTGAAACATCTTAATCATGGAAGGGAG GGT F1-SD2dCas9 ACATCTTAATCATGCAAAGGAGGTGTTTTAATGGATAAGAAATACTCAATAGGC TTAGC F2-SD2dCas9 AGCTCGAGATGAAAGCAATTTTCGTACTGAAACATCTTAATCATGCAAAGGAG GTG R-Not-dCas9 GCGCGGCCGCTTATTAGTGG F-RT-sfGFP TGCTACAAACGGAAAACTCA R-RT-sfGFP GCGTTCCTGTACATAACCTT F-RT-folE ACCTCGCTTGGTTGATTGGG R-RT-folE CGTGTTTGTTTGCGTCCCTG F-RT-ribF TCCACATCCAAGTGTCGCAT R-RT-ribF CCTGTGGTGATAAACCCGCT F-RT-16srRNA TATGTCCCGAGCGTTATCCG R-RT-16srRNA TCTACGCATTCCACCGCTAC F-Erm AATCTAGAGCATTGACGTCAGATACTGCA R-Erm TTAGGCCTCTGCAGGAGCTTTGGCTAA F1-v4-ribD ATAATGAGTATAACCAGAAGAAAGGATAGAAAAAATGGATGATTTAACATATAT GGCATTAGCA F2-v4-ribD GTGTTTTTAGCCTGAAGTGTTATAATGAGTATAACCAGAAGAAAGGATAGAAA F3-v4-ribD GACTGCAGGAGAACAGTTTAAAGTTAATAGGTGTTTTTAGCCTGAAGTGTTATA ATGA R-ribH TTCTGCAGTCAGTGGTGGTGGTGGTGGTGAGACAGTTGTTTTATGTCATCATAT AAACTT

Figure S1 .

 S1 Figure S1. Schematic of synthetic guide RNAs target sequence used in this study. NT 


Table 1 . Bacterial strains and plasmids used in this study.

 1 
	Plasmid	Description	Reference
	Strains		
	XL 1-Blue	recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´	Stratagene a
		proAB lacIqZDM15 Tn10 (Tetr)]	
	L. citreum CB2567 Wild type	[38]
	JW001	L. citreum CB2567, ΔldhD::P710-sfGFP-Cm R	This study
	Plasmids		
	pCB4270	L. citreum-E. coli shuttle vector, P 710 , Amp R , Cm R	[19]
	pCB4270-Em	L. citreum-E. coli shuttle vector, P 710 , Amp R , Em R	This study
	pCB4270-sfGFP	Monocistronic, P710, sfGFP, Amp R , Cm R	[19]
	pCB4270V4	L. citreum-E. coli shuttle vector, P 710V4 , Amp R , Cm R	This study
	pCB4270V4-Em	L. citreum-E. coli shuttle vector, P 710V4 , Amp R , Em R	This study
	pdCas9	Plasmid carrying SpdCas9 catalytic site mutant	Addgene, USA
	pgRNA-bacteria	Plasmid carrying customized tracrRNA for bacterial gene	Addgene, USA
		knock-down	
	pSOS95	Acetone operon, repL gene, ColE1 origin, Amp R , MLS R ,	[39]
		Em R	
	pHldhU-sfGFP	pCB4270-sfGFP derivative containing 0.8 kb homologous	This study
		region BamHI upstream fragment of ldhD	
	pH-InsfGFP	pCB4270ldhU-sfGFP derivative suicide vector containing	This study
		0.8 kb homologous region BamHI upstream fragment of	
		ldhD and 1 kb homologous region PstI internal fragment of	
		ldhD, Amp R , Cm R	
	pGFP-sgR-Em	pCB4270-Em derivative carrying P 710V4 , sfGFP targeting	This study
		sgRNA, Amp R , Em R	
	pD1-Em	pCB4270V4-Em derivative carrying bicistronic, P 710V4 ,	This study
		eSD2, SpdCas9, His tag, Amp R , Em R	
	pD2-Em	pCB4270V4-Em derivative carrying bicistronic, P 710V4 ,	This study
		SD2, SpdCas9, His tag, Amp R , Em R	
	pD3-Em	pCB4270-Em derivative carrying bicistronic, P 710 , eSD2,	This study
		SpdCas9, His tag, Amp R , Em R	
	pD4-Em	pCB4270-Em derivative carrying bicistronic, P 710 , SD2,	This study
		SpdCas9, His tag, Amp R , Em R	
	pD1	pCB4270V4 derivative carrying bicistronic, P 710V4 , eSD2,	This study
		SpdCas9, His tag, Amp R , Cm R	
	pD2	pCB4270V4 derivative carrying bicistronic, P 710V4 , SD2,	This study
		SpdCas9, His tag, Amp R , Cm R	
	pD3	pCB4270 derivative carrying bicistronic, P 710 , eSD2,	This study
		SpdCas9, His tag, Amp R , Cm R	
	pD4	pCB4270 derivative carrying bicistronic, P 710 , SD2,	This study
		SpdCas9, His tag, Amp R , Cm R	
	pGFP-sgR-D1-Em pD1-Em derivative carrying bicistronic, P 710V4 , eSD2,	This study
		SpdCas9, His tag, P 710V4 , sfGFP targeting sgRNA, Amp R ,	
		Em R	
	pGFP-sgR-D2-Em pD2-Em derivative carrying bicistronic, P 710V4 , SD2,	This study
		SpdCas9, His tag, P 710V4 , sfGFP targeting sgRNA, Amp R ,	
		Em R	

Table S2 : Oligonucleotides primers used in this study Primer Sequence (5' to 3')

 S2 

Table S3 . sgRNA used in this study

 S3 
	sgRNA name	Target location	Spacer sequence	PAM sequence
			(5' to 3')	(5' to 3')
	GFP-sgRNA	CDS of sfGFP	AATTCTTGTTGAATTAGATG	CCC
	folE-sgRNA	CDS of folE	ATATTTGACGCCCTGCACGA	CCC
	ribF-sgRNA	CDS of ribF	GGCTGTTTGAAATTTTGAAT	CCA

Supplementary Information


Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboflavin Biosynthesis

Jaewoo Son 1 , Seung Hoon Jang 1 , Ji Won Cha 1 and Ki Jun Jeong 1,2 *