Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4

Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeats; sgRNA, single-guide RNA; tracrRNA, trans-activating CRISPR RNA; crRNA, CRISPR RNA; Cas, CRISPR-associated; PAM, Protospacer-Adjacent Motif; qPCR, quantitative (real time) PCR; RMG, Rich medium with glucose.

Figures & data

Table 1. Primers used for construction of Cas9-expression plasmid and amplification of templates for in vitro transcription.

Primer	Sequence (5′–3′)^*	Description
cas9-F	ATATATGGAGTAAGCAATGGATAAGAAATACTC	Cas9 gene of S. pyogenes CICC10464
cas9-R	CCCACTGCAAGCTACCTTCAGTCACCTCCTAG
Pcas9-F	CTAGGAGGTGACTGAAGGTAGCTTGCAGTGGG	The vector DNA fragment of pSUZM2a
Pcas9-R	GAGTATTTCTTATCCATTGCTTACTCCATATATG
upp1F	CTATGTTGTGCCAATTCTGCGTGC	First target position of upp gene
upp1R	AAACGCACGCAGAATTGGCACAACA
upp2F	CTATGTACCGTAATGAAGAAACGC	Second target position of upp gene
upp2R	AAACGCGTTTCTTCATTACGGTACA
ZM4023F1	CTATGGCCTTATCGCTTAAGCCCT	First target position of replicase gene
ZM4023R1	AAACAGGGCTTAAGCGATAAGGCCA
ZM4023F3	CTATGGTATCCTCCTTAGGTGTTT	Second target position of replicase gene
ZM4023R3	AAACAAACACCTAAGGAGGATACCA
transtempF	GCCACCTCTGACTTGA	amplification of templates for transcription
transtempR	CAAAAAACCCCTCAAGACCCG

^* The sequences underlined are target positions.

Table 2. Primers used for PCR assays to identify plasmids.

Primer	Sequence (5′–3′)^*	Description
pZM4GF	GATGCTGTTTCGGATGGTCGAC	Forward detection primer for five native plasmids
pZM401R	GTCTTGACGGTAACCGCGAG	Reverse detection primer for pZZM401 (GI:8764733 for ZZM4_0043)
pZM402R	GATCAGGACGATAACCGCGAG	Reverse detection primer for pZZM402 (GI:8764764 for ZZM4_0071)
pZM403R	GGTCGGGACG ATAACCACG	Reverse detection primer for pZZM403 (GI:8764795 for ZZM4_0103)
pZM404R	ATCGGGGCGATAACCACGAG	Reverse detection primer for pZZM404 (GI:8764817 for ZZM4_0122)
pZM405R	CTGATCAGGCCTTAGGTTCAGG	Reverse detection primer for pZZM405 (GI:8764868 for ZZM4_0166)
upptestF	TAATCTTCTTTCATAACCATCTG	Detection primers for upp gene mutation (GI:481024210)
upptestR	CTAAGCGGGATTGTCTGT
01-qF	GAGCCAACTTTGATGCG CGAGAATGCCACTGACG	qPCR primers for pZZM401 (GI: 8764708 for ZZM4_0018)
01-qR
02-qF	AAATCGGTCGTCTAAC ACACCACCTGAACAAA	qPCR primers for pZZM402 (GI: 8764750 for ZZM4_0057)
02-qR
03-qF	CCGTACATCTAAATCCG TTCTCGCAAGACCAAT	qPCR primers for pZZM403 (GI: 8764799 for ZZM4_0107)
03-qR
04-qF	GAACGGTGGGTGTCTC AGCCTCAGCCCTTGTA	qPCR primers for pZZM404 (GI: 8764823 for ZZM4_0131)
04-qR
pdcqF	CAAAGTTGCCGTCCTC GGTGCCGATGTAATGC	qPCR primers for pyruvate decarboxylase gene (GI: 56543830 for ZMO1360)
pdcqR

^* The 700-bp fragment of the each replicase gene of the native plasmids was generated using the primers pZM4GF/pZM401R, pZM4GF/pZM402R, pZM4GF/pZM403R, pZM4GF/pZM404R, and pZM4GF/pZM405R.

Fig. 1. Physical maps of pSUZM2a-Cas9 and pUC-T7sgRNA.

Note: (A) The physical map of pSUZM2a-Cas9. (B) The physical map of pUC-T7sgRNA. (C) Restriction enzyme digestion of the expression plasmid pSUZM2a-Cas9. The plasmids were digested with KpnI, lane 1: pSUZM2a; lane 2: pSUZM2a-Cas9; M, λ-EcoT14 I DNA marker.

Fig. 2. Generation of region-specific deletions in the upp gene using the CRISPR/Cas9 system.

Note: (A) PCR identification of upp gene deletion mutants in E. coli DH5α, lane 1: mutant; lane 2: wild type; M, DL1000 DNA marker. (B) DNA sequence analysis of the upp gene from the mutant and wild type using DNAman, the box for DNA fragments of two target sites.

Fig. 3. Plasmid-specific PCR and quantitative PCR analysis of ZM4-∆403 and ZM4.

Note: (A) The multiple nucleotide sequence alignments of the five replicase genes from the five native ZM4 plasmids using DNAMAN software. (B) Two target positions of the replicase genes of pZZM402 and pZZM403 plasmids in ZM4. Left: second target position of replicase gene; right: first target position of replicase gene. (C) PCR analysis of ZM4-∆403 (Lanes 1–5) and ZM4 (lanes 6–10). Lanes 1 and 6: pZZM401; lanes 2 and 7: pZZM402; lanes 3 and 8: pZZM403; lanes 4 and 9: pZZM404; lanes 5 and 10: pdc. (D) Quantitative PCR analysis of plasmid copy number in ZM4-∆403 (left) and ZM4 (right).

Fig. 4. Growth curve of ZM4-∆403 and ZM4 under normal growth conditions.