Journal: Microbial Cell Factories

Article Title: Gene repression via multiplex gRNA strategy in Y. lipolytica

doi: 10.1186/s12934-018-0909-8

Figure Lengend Snippet: Schematic diagram of gene repression via multiplex CRISPRi system in Y. lipolytica . The combination of four different repressors (dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB) and ten gRNAs that bind to different regions of gfp gene were employed in Y. lipolytica . But the results indicated that there was no clear correlation between the repression efficiency and targeting sites (left). As extra target sites screening often mandated a significant investment of time and effort, a strategy via one-step Golden-brick assembly of multiplex gRNAs was established for high-efficiency and tunable perturbation of gene expression in Y. lipolytica (right)

Article Snippet: The fragments of dCpf1 (D917A) derived from Francisella novicida U112 (NC_00860 1) [ ], Cas9 and dCas9 (D10A and H841A) derived from Streptococcus pyogenes [ ], dCas9 - KRAB [ ], dCpf1 - KRAB , sfGFP [ , ], VioA, VioB, VioE [ ], and vector assembled modules of pMCSCen1 [ ], PMCS-Multi, JLPC/N-n and JLRC/N-n were codon optimized and synthesized by GenScript (Nanjing China). gRNA oligos were synthesized by Genewiz (Suzhou, China).

Techniques: Multiplex Assay, Expressing

Journal: Microbial Cell Factories

Article Title: Gene repression via multiplex gRNA strategy in Y. lipolytica

doi: 10.1186/s12934-018-0909-8

Figure Lengend Snippet: Repression of gfp in Y. lipolytica by dCpf1 and dCas9. a The dCpf1 and dCas9 expression system. The dCpf1 contains mutations of the RuvC1 nuclease domains while the dCas9 contains mutations of both RuvC1 and HNH nuclease domains. b Schematic of RNA polymerase III promoter used in this study and placement of gRNA protospacers on the target gfp gene. SCR1′-tRNA Gly is the synthetic RNA polymerase III promoter. gRNA is single guide RNAs. polyT is a string of eight thymines, which serves as an RNA polymerase III terminator. Six gRNAs bind to either the non-template DNA strand or the template DNA strand of ORF and four gRNAs bind to different regions around the promoter. c Microscopic images of the interfered strains with gfp gene by dCpf1. d Microscopic images of the interfered strains with gfp gene by dCas9. e CRISPRi repression of gfp with dCpf1 complexed with ten gRNAs targeting different regions. The control (g0) shows fluorescence of the cells with dCpf1 protein but without the gRNA. f CRISPRi repression of gfp with dCas9 complexed with ten gRNAs targeting different regions. The control (g0) shows fluorescence of the cells with dCas9 protein but without the gRNA. g Characterization of the gfp gene’s expression level of each strain interfered by dCpf1. h Characterization of the gfp gene’s expression level of each strain interfered by dCas9. The error bars (mean ± SD) were derived from triplicate experiments for each strain

Article Snippet: The fragments of dCpf1 (D917A) derived from Francisella novicida U112 (NC_00860 1) [ ], Cas9 and dCas9 (D10A and H841A) derived from Streptococcus pyogenes [ ], dCas9 - KRAB [ ], dCpf1 - KRAB , sfGFP [ , ], VioA, VioB, VioE [ ], and vector assembled modules of pMCSCen1 [ ], PMCS-Multi, JLPC/N-n and JLRC/N-n were codon optimized and synthesized by GenScript (Nanjing China). gRNA oligos were synthesized by Genewiz (Suzhou, China).

Techniques: Expressing, Fluorescence, Derivative Assay

Journal: Microbial Cell Factories

Article Title: Gene repression via multiplex gRNA strategy in Y. lipolytica

doi: 10.1186/s12934-018-0909-8

Figure Lengend Snippet: Repression of gfp in Y. lipolytica by dCpf1-KRAB and dCas9-KRAB. a Schematic of dCpf1 fused to repressor domain KRAB and the dCpf1-KRAB expression system. b Schematic of dCas9 fused to repressor domain KRAB and the dCas9-KRAB expression system. c CRISPRi repression of gfp with dCpf1-KRAB complexed with ten gRNAs targeting different regions. The control (g0) shows fluorescence of the cells with dCpf1-KRAB protein but without the gRNA. d CRISPRi repression of gfp with dCas9-KRAB complexed with ten gRNAs targeting different regions. The control (g0) shows fluorescence of the cells with dCas9-KRAB protein but without the gRNA. The error bars (mean ± SD) were derived from triplicate experiments for each strain

Article Snippet: The fragments of dCpf1 (D917A) derived from Francisella novicida U112 (NC_00860 1) [ ], Cas9 and dCas9 (D10A and H841A) derived from Streptococcus pyogenes [ ], dCas9 - KRAB [ ], dCpf1 - KRAB , sfGFP [ , ], VioA, VioB, VioE [ ], and vector assembled modules of pMCSCen1 [ ], PMCS-Multi, JLPC/N-n and JLRC/N-n were codon optimized and synthesized by GenScript (Nanjing China). gRNA oligos were synthesized by Genewiz (Suzhou, China).

Techniques: Expressing, Fluorescence, Derivative Assay

Journal: Microbial Cell Factories

Article Title: Gene repression via multiplex gRNA strategy in Y. lipolytica

doi: 10.1186/s12934-018-0909-8

Figure Lengend Snippet: An overview of the Golden-Brick assembly protocol. Multiplex CRISPRi system contains two main parts, one is JLPC/N-n (or JLRC/N-n) plasmid containing gRNA secretion cassette enabling spacers to be ligated into, the other is PMCS-Multi-CRI vector (classified as dCpf1-Multi and dCas9-Multi) containing the gblock of ‘A’ overhang and ‘T’ overhang enabling to assemble various gRNA secretion cassettes. After being released, these gRNA secretion cassettes were assembled with dCpf1-Multi or dCas9-Multi vector in one step. JLPN-n and JLRN-n plasmids were constructed by replacing synthetic hybrid promoter SCR1′-tRNA Gly (JLPC-n and JLRC-n gRNA expression promoter region) with SNR52′-tRNA Gly

Article Snippet: The fragments of dCpf1 (D917A) derived from Francisella novicida U112 (NC_00860 1) [ ], Cas9 and dCas9 (D10A and H841A) derived from Streptococcus pyogenes [ ], dCas9 - KRAB [ ], dCpf1 - KRAB , sfGFP [ , ], VioA, VioB, VioE [ ], and vector assembled modules of pMCSCen1 [ ], PMCS-Multi, JLPC/N-n and JLRC/N-n were codon optimized and synthesized by GenScript (Nanjing China). gRNA oligos were synthesized by Genewiz (Suzhou, China).

Techniques: Multiplex Assay, Plasmid Preparation, Construct, Expressing

Journal: Microbial Cell Factories

Article Title: Gene repression via multiplex gRNA strategy in Y. lipolytica

doi: 10.1186/s12934-018-0909-8

Figure Lengend Snippet: Repression of gfp via Multiplex CRISPRi system in Y. lipolytica . Regulation of gfp expression by dCpf1-Multi and dCas9-Multi system combined with multiplex gRNA targets. a Repression of gfp by dCpf1-Multi and dCas9-Multi system complexed with single gRNA, double gRNAs and triple gRNAs expressed by SCR1′-tRNA Gly or SNR52′-tRNA Gly promoter. b Characterization of the gfp gene’s expression level of each strain interfered by dCpf1-Multi and dCas9-Multi system. “+” means possess and “−” means not possess. The error bars (mean ± SD) were derived from triplicate experiments for each strain

Article Snippet: The fragments of dCpf1 (D917A) derived from Francisella novicida U112 (NC_00860 1) [ ], Cas9 and dCas9 (D10A and H841A) derived from Streptococcus pyogenes [ ], dCas9 - KRAB [ ], dCpf1 - KRAB , sfGFP [ , ], VioA, VioB, VioE [ ], and vector assembled modules of pMCSCen1 [ ], PMCS-Multi, JLPC/N-n and JLRC/N-n were codon optimized and synthesized by GenScript (Nanjing China). gRNA oligos were synthesized by Genewiz (Suzhou, China).

Techniques: Multiplex Assay, Expressing, Derivative Assay

Journal: Microbial Cell Factories

Article Title: Gene repression via multiplex gRNA strategy in Y. lipolytica

doi: 10.1186/s12934-018-0909-8

Figure Lengend Snippet: Multiplex gene interference in Y. lipolytica . a Heterogenous biosynthesis pathway for protodeoxy-violaceinic acid (PVA) production in Y. lipolytica . b Schematic of the plasmid targeting vioA , vioB and vioE simultaneously. Schematic of plasmid targeting both gfp and vioE . c The PVA relative absorbance of strains interfered by dCpf1-Multi and dCas9-Multi system. d The fluorescence and the PVA relative absorbance of strains interfered by dCas9-Multi system. “+” means possess and “−” means not possess. The error bars (mean ± SD) were derived from triplicate experiments for each strain

Article Snippet: The fragments of dCpf1 (D917A) derived from Francisella novicida U112 (NC_00860 1) [ ], Cas9 and dCas9 (D10A and H841A) derived from Streptococcus pyogenes [ ], dCas9 - KRAB [ ], dCpf1 - KRAB , sfGFP [ , ], VioA, VioB, VioE [ ], and vector assembled modules of pMCSCen1 [ ], PMCS-Multi, JLPC/N-n and JLRC/N-n were codon optimized and synthesized by GenScript (Nanjing China). gRNA oligos were synthesized by Genewiz (Suzhou, China).

Techniques: Multiplex Assay, Plasmid Preparation, Fluorescence, Derivative Assay