目的:探索CRISPR干扰(CRISPR interference,C R I S P R i)能否实现在体抑制肝脏m i R-122表达.方法:针对m i R-1 2 2启动子区设计sg RNA(sg T1和sg T2),并分别将其与无DNA切割活性仅保留识别活性的d Cas9-KRAB载体通过尾静脉流体力学...目的:探索CRISPR干扰(CRISPR interference,C R I S P R i)能否实现在体抑制肝脏m i R-122表达.方法:针对m i R-1 2 2启动子区设计sg RNA(sg T1和sg T2),并分别将其与无DNA切割活性仅保留识别活性的d Cas9-KRAB载体通过尾静脉流体力学法注射到8-10 wk龄小鼠,注射1、2、4 wk后通过实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)方法检测肝脏mmu-mi R-122的表达;设计不同的sg RNA浓度梯度,探索CRISPRi在体抑制肝脏mi R-122表达是否存在剂量依赖性;通过q RT-PCR及Western blot方法检测肝脏mi R-122靶分子HOMX1和Cyclin G1的表达变化.结果:在注射1 wk和2 wk后,sg T1介导的C R I S P R i在体抑制肝脏m i R-122的表达水平分别为23%(P<0.05)和16%(P<0.05);随sg RNA的剂量升高,肝脏mi R-122表达降低,当lenti Guide-Puro-sg T1质粒为120?g时,可将mi R-122的表达抑制约30%;CRIPSRi在体抑制肝脏mi R-122表达的同时,上调了mi R-122下游靶分子HMOX1和Cyclin G1的表达.结论:本研究利用CRISPRi实现了在体抑制肝脏mi R-122的表达,为抗丙型肝炎病毒(hepatitis C virus)的在体治疗提供了新的策略.展开更多
Lactate is an important monomer for the synthesis of poly-lactate(PLA),which is a substitute for the petrochemical plastics.To achieve the goal of high lactate titer,rate,and yield for commercial production,efficient ...Lactate is an important monomer for the synthesis of poly-lactate(PLA),which is a substitute for the petrochemical plastics.To achieve the goal of high lactate titer,rate,and yield for commercial production,efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and tolerance.In this study,an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence,which could be the mutant strains with increased d-lactate production.Then,LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference(CRISPRi)library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole genome.Specifically,two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting(FACS),respectively.Two genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production,further knockout of ZMO1323 and ZMO1530 resulted in a 15%and 21%increase of d-lactate production,respectively.This work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in Z.mobilis,but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.展开更多
Formic acid is one of the main weak acids in lignocellulosic hydrolysates that is known to be inhibitory to yeast growth even at low concentrations.In this study,we employed a CRISPR interference(CRISPRi)strain librar...Formic acid is one of the main weak acids in lignocellulosic hydrolysates that is known to be inhibitory to yeast growth even at low concentrations.In this study,we employed a CRISPR interference(CRISPRi)strain library comprising>9000 strains encompassing>98%of all essential and respiratory growth-essential genes,to study formic acid tolerance in Saccharomyces cerevisiae.To provide quantitative growth estimates on formic acid toler-ance,the strains were screened individually on solid medium supplemented with 140 mM formic acid using the Scan-o-Matic platform.Selected resistant and sensitive strains were characterized in liquid medium supplemented with formic acid and in synthetic hydrolysate medium containing a combination of inhibitors.Strains with gR-NAs targeting genes associated with chromatin remodeling were significantly enriched for strains showing formic acid tolerance.In line with earlier findings on acetic acid tolerance,we found genes encoding proteins involved in intracellular vesicle transport enriched among formic acid sensitive strains.The growth of the strains in syn-thetic hydrolysate medium followed the same trend as when screened in medium supplemented with formic acid.Strains sensitive to formic acid had decreased growth in the synthetic hydrolysate and all strains that had im-proved growth in the presence of formic acid also grew better in the hydrolysate medium.Systematic analysis of CRISPRi strains allowed identification of genes involved in tolerance mechanisms and provided novel engineering targets for bioengineering strains with increased resistance to inhibitors in lignocellulosic hydrolysates.展开更多
CRISPR interference(CRISPRi)has been developed and widely used for gene repression in various hosts.Here we report an improved CRISPRi system in Pichia pastoris by fusing dCas9 with endogenous transcriptional represso...CRISPR interference(CRISPRi)has been developed and widely used for gene repression in various hosts.Here we report an improved CRISPRi system in Pichia pastoris by fusing dCas9 with endogenous transcriptional repressor domains.The CRISPRi system shows strong repression of eGFP,with the highest efficiency of 85%.Repression of native genes is demonstrated by targeting AOX1 promoter.AOX1 is efficiently repressed and the mutant strains show much slower growth in methanol medium.Effects of gRNA expression and processing on CRISPRi efficiency is also investigated.It is found that gRNA processing by HH/HDV ribozymes or Csy4 endoribonuclease generating clean gRNA is critical to achieve strong repression,and Csy4 cleavage shows higher repression efficiency.However,gRNA expression using native tRNA transcription and processing systems results in relatively weaker repression of eGFP.By expression of two gRNAs targeting promoters of eGFP and AOX1 in an array together with Cys4 recognition sites,both genes can be repressed simultaneously.Cys4-mediated gRNA array processing is further applied to repress fatty acyl-CoA synthetase genes(FAA1 and FAA2).Both genes are efficiently repressed,demonstrating that Cys4 endoribonuclease has the ability to cleave gRNAs array and can be can be used for multiplexed gene repression in P.pastoris.展开更多
The production ofβ-amyrin in Saccharomyces cerevisiae is still low due to the inability of effectively regulating the endogenous metabolic pathway for competitive synthesis ofβ-amyrin precursors.In this study,we foc...The production ofβ-amyrin in Saccharomyces cerevisiae is still low due to the inability of effectively regulating the endogenous metabolic pathway for competitive synthesis ofβ-amyrin precursors.In this study,we focused on two branches ofβ-amyrin synthetics pathway that consumeβ-amyrin precursors(2,3-oxidosqualene and cytosolic acetyl-CoA)and regulated related genes(ADH1,ADH4,ADH5,ADH6,CIT2,MLS2 and ERG7).We developed a CRISPRi method by constructing a multi-gRNA plasmid to down-regulate the seven genes simultaneously,which is reported for the first time in S.cerevisiae.The average transcription inhibition efficiency of the seven genes reached as high as 75.5%.Furthermore,by optimizing the fermentation condition(including pH,inoculum size,initial glucose concentration and feed of glucose or ethanol)and increasing extracellular transportation via supplying methyl-β-cyclodextrin,β-amyrin concentration of engineered strain SGibSdCg increased by 44.3%compared with the parent strain SGib,achieving 156.7 mg/L which was the highest concentration ofβ-amyrin reported in yeast.The one-step down-regulation of multiple genes using CRISPRi showed high efficiency and promising future in improving the yields of natural products.展开更多
Itaconic acid(ITA),an effective alternative fossil fuel,derives from the bypass pathway of the tricarboxylic acid(TCA)cycle.Therefore,the imbalance of metabolic flux between TCA cycle and ITA biosynthetic pathway seri...Itaconic acid(ITA),an effective alternative fossil fuel,derives from the bypass pathway of the tricarboxylic acid(TCA)cycle.Therefore,the imbalance of metabolic flux between TCA cycle and ITA biosynthetic pathway seriously limits the production of ITA.The optimization of flux distribution between biomass and production has the potential to the productivity of ITA.Based on the previously constructed strain Escherichia coli MG1655Δ1-SAS-3(ITA titer:1.87 g/L),a CRISPRi-mediated self-inducible system(CiMS),which contained a responsive module based on the ITA biosensor YpItcR/Pccl and a regulative CRISPRi-mediated interferential module,was developed to regulate the flux of the TCA cycle and to enhance the capacity of the strain to produce ITA.First,a higher ITA-yielding strain,Δ4-Prmd-SAS-3(ITA titer:3.20 g/L),derived fromΔ1-SAS-3,was constructed by replacing the promoter PJ23100,for the expression of ITA synthesis genes,with Prmd and knocking out the three bypass genes poxB,pflB,and ldhA.Subsequently,the CiMS was used to inhibit the expression of key genes icd,pykA,and sucCD to dynamically balance the metabolic flux between TCA cycle and ITA biosynthetic pathway during the ITA production stage.The constructed strainΔ4-Prmd-SAS-3 under the dynamic regulation of the CiMS,showed a 23%increase in the ITA titer,which reached 3.93 g/L.This study indicated that CiMS was a practical strategy to dynamically and precisely regulated the metabolic flux in microbial cell factories.展开更多
Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the effici...Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference(CRISPRi) and Natronobacterium gregoryi Argonaute(NgAgo) interference(NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase(PNKP), DNA ligase IV(LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted(P<0.05) the efficiency of HR to 1.85-and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase(P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed(P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve(P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9(dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.展开更多
文摘目的:探索CRISPR干扰(CRISPR interference,C R I S P R i)能否实现在体抑制肝脏m i R-122表达.方法:针对m i R-1 2 2启动子区设计sg RNA(sg T1和sg T2),并分别将其与无DNA切割活性仅保留识别活性的d Cas9-KRAB载体通过尾静脉流体力学法注射到8-10 wk龄小鼠,注射1、2、4 wk后通过实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)方法检测肝脏mmu-mi R-122的表达;设计不同的sg RNA浓度梯度,探索CRISPRi在体抑制肝脏mi R-122表达是否存在剂量依赖性;通过q RT-PCR及Western blot方法检测肝脏mi R-122靶分子HOMX1和Cyclin G1的表达变化.结果:在注射1 wk和2 wk后,sg T1介导的C R I S P R i在体抑制肝脏m i R-122的表达水平分别为23%(P<0.05)和16%(P<0.05);随sg RNA的剂量升高,肝脏mi R-122表达降低,当lenti Guide-Puro-sg T1质粒为120?g时,可将mi R-122的表达抑制约30%;CRIPSRi在体抑制肝脏mi R-122表达的同时,上调了mi R-122下游靶分子HMOX1和Cyclin G1的表达.结论:本研究利用CRISPRi实现了在体抑制肝脏mi R-122的表达,为抗丙型肝炎病毒(hepatitis C virus)的在体治疗提供了新的策略.
基金This work was supported by the National Key Research and Development Program of China(2022YFA0911800)National Natural Science Foundation of China(CN)(21978071)+2 种基金the Key Science and Technology Innovation Project of Hubei Province(2021BAD001)the Innovation Base for Introducing Talents of Discipline of Hubei Province(2019BJH021)We also acknowledge the support from the State Key Laboratory of Biocatalysis and Enzyme Engineering.
文摘Lactate is an important monomer for the synthesis of poly-lactate(PLA),which is a substitute for the petrochemical plastics.To achieve the goal of high lactate titer,rate,and yield for commercial production,efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and tolerance.In this study,an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence,which could be the mutant strains with increased d-lactate production.Then,LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference(CRISPRi)library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole genome.Specifically,two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting(FACS),respectively.Two genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production,further knockout of ZMO1323 and ZMO1530 resulted in a 15%and 21%increase of d-lactate production,respectively.This work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in Z.mobilis,but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.
基金The authors would like to thank Cecilia Trivellin for providing the original R code for the analysis of the growth curves(available at https://github.com/cectri/Quantification-of-microbial-robustness.git)as well as Luca Torello Pianale for support in strain analysis.We acknowledge the Novo Nordisk Foundation(NF19OC0057685)The Swedish Research Council(Dnr 2018-04713)and the Hasselblad Foundation for financial support.
文摘Formic acid is one of the main weak acids in lignocellulosic hydrolysates that is known to be inhibitory to yeast growth even at low concentrations.In this study,we employed a CRISPR interference(CRISPRi)strain library comprising>9000 strains encompassing>98%of all essential and respiratory growth-essential genes,to study formic acid tolerance in Saccharomyces cerevisiae.To provide quantitative growth estimates on formic acid toler-ance,the strains were screened individually on solid medium supplemented with 140 mM formic acid using the Scan-o-Matic platform.Selected resistant and sensitive strains were characterized in liquid medium supplemented with formic acid and in synthetic hydrolysate medium containing a combination of inhibitors.Strains with gR-NAs targeting genes associated with chromatin remodeling were significantly enriched for strains showing formic acid tolerance.In line with earlier findings on acetic acid tolerance,we found genes encoding proteins involved in intracellular vesicle transport enriched among formic acid sensitive strains.The growth of the strains in syn-thetic hydrolysate medium followed the same trend as when screened in medium supplemented with formic acid.Strains sensitive to formic acid had decreased growth in the synthetic hydrolysate and all strains that had im-proved growth in the presence of formic acid also grew better in the hydrolysate medium.Systematic analysis of CRISPRi strains allowed identification of genes involved in tolerance mechanisms and provided novel engineering targets for bioengineering strains with increased resistance to inhibitors in lignocellulosic hydrolysates.
基金National Key Research and Development Program of China(2021YFC2103500)Dalian Institute of Chemical Physics Innovation Program(DICP I202111).
文摘CRISPR interference(CRISPRi)has been developed and widely used for gene repression in various hosts.Here we report an improved CRISPRi system in Pichia pastoris by fusing dCas9 with endogenous transcriptional repressor domains.The CRISPRi system shows strong repression of eGFP,with the highest efficiency of 85%.Repression of native genes is demonstrated by targeting AOX1 promoter.AOX1 is efficiently repressed and the mutant strains show much slower growth in methanol medium.Effects of gRNA expression and processing on CRISPRi efficiency is also investigated.It is found that gRNA processing by HH/HDV ribozymes or Csy4 endoribonuclease generating clean gRNA is critical to achieve strong repression,and Csy4 cleavage shows higher repression efficiency.However,gRNA expression using native tRNA transcription and processing systems results in relatively weaker repression of eGFP.By expression of two gRNAs targeting promoters of eGFP and AOX1 in an array together with Cys4 recognition sites,both genes can be repressed simultaneously.Cys4-mediated gRNA array processing is further applied to repress fatty acyl-CoA synthetase genes(FAA1 and FAA2).Both genes are efficiently repressed,demonstrating that Cys4 endoribonuclease has the ability to cleave gRNAs array and can be can be used for multiplexed gene repression in P.pastoris.
基金the funds support from National Natural Science Foundation of China(21576027,21425624,21736002,21566032).
文摘The production ofβ-amyrin in Saccharomyces cerevisiae is still low due to the inability of effectively regulating the endogenous metabolic pathway for competitive synthesis ofβ-amyrin precursors.In this study,we focused on two branches ofβ-amyrin synthetics pathway that consumeβ-amyrin precursors(2,3-oxidosqualene and cytosolic acetyl-CoA)and regulated related genes(ADH1,ADH4,ADH5,ADH6,CIT2,MLS2 and ERG7).We developed a CRISPRi method by constructing a multi-gRNA plasmid to down-regulate the seven genes simultaneously,which is reported for the first time in S.cerevisiae.The average transcription inhibition efficiency of the seven genes reached as high as 75.5%.Furthermore,by optimizing the fermentation condition(including pH,inoculum size,initial glucose concentration and feed of glucose or ethanol)and increasing extracellular transportation via supplying methyl-β-cyclodextrin,β-amyrin concentration of engineered strain SGibSdCg increased by 44.3%compared with the parent strain SGib,achieving 156.7 mg/L which was the highest concentration ofβ-amyrin reported in yeast.The one-step down-regulation of multiple genes using CRISPRi showed high efficiency and promising future in improving the yields of natural products.
基金supported by the National Natural Science Foundation of China(No.21778018)Research Program of State Key Laboratory of Bioreactor Engineering and the grant from the National Key Research and Development Program of China(2021YFC2100300)the Chinese Plastic Surgery Foundation(No.2020M671021).
文摘Itaconic acid(ITA),an effective alternative fossil fuel,derives from the bypass pathway of the tricarboxylic acid(TCA)cycle.Therefore,the imbalance of metabolic flux between TCA cycle and ITA biosynthetic pathway seriously limits the production of ITA.The optimization of flux distribution between biomass and production has the potential to the productivity of ITA.Based on the previously constructed strain Escherichia coli MG1655Δ1-SAS-3(ITA titer:1.87 g/L),a CRISPRi-mediated self-inducible system(CiMS),which contained a responsive module based on the ITA biosensor YpItcR/Pccl and a regulative CRISPRi-mediated interferential module,was developed to regulate the flux of the TCA cycle and to enhance the capacity of the strain to produce ITA.First,a higher ITA-yielding strain,Δ4-Prmd-SAS-3(ITA titer:3.20 g/L),derived fromΔ1-SAS-3,was constructed by replacing the promoter PJ23100,for the expression of ITA synthesis genes,with Prmd and knocking out the three bypass genes poxB,pflB,and ldhA.Subsequently,the CiMS was used to inhibit the expression of key genes icd,pykA,and sucCD to dynamically balance the metabolic flux between TCA cycle and ITA biosynthetic pathway during the ITA production stage.The constructed strainΔ4-Prmd-SAS-3 under the dynamic regulation of the CiMS,showed a 23%increase in the ITA titer,which reached 3.93 g/L.This study indicated that CiMS was a practical strategy to dynamically and precisely regulated the metabolic flux in microbial cell factories.
基金supported by the National Science and Technology Major Project for Breeding of New Transgenic Organisms, China (2016ZX08006002)the Guangdong Province "Flying Sail Program" Postdoctoral Foundation, China (2016)
文摘Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference(CRISPRi) and Natronobacterium gregoryi Argonaute(NgAgo) interference(NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase(PNKP), DNA ligase IV(LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted(P<0.05) the efficiency of HR to 1.85-and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase(P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed(P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve(P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9(dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.
