Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inac...Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools.In this work, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs(PAMs) based on bioinformatics analysis combined with a plasmid interference assay in S. spinosa. By delivering editing plasmids containing a designed mini CRISPR array(repeat+self-targeting spacer+repeat) and repair templates, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the restriction-modification(RM) systems was systematically disarmed. We showed that by disarming one type I RM system(encoded by A8926_1903/1904/1905) and two type Ⅱ RM systems(encoded by A8926_1725/1726 and A8926_2652/2653) simultaneously, the transformation efficiency of the replicative and integrative plasmids(pSP01 and pSI01) was increased by approximately 3.9-fold and 4.2-fold, respectively. Using the engineered strain with simultaneous knock-out of these three RM genes as the starting strain, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster(BGC) as well as gene insertion at high efficiency. Collectively, we developed a reliable and highly efficient genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.展开更多
目的探讨丹红注射液联合左西孟旦对脓毒症心肌病患者N-末端B型脑钠肽前体(NT-proBNP)、肌钙蛋白I(cTnⅠ)水平及血流动力学的影响。方法研究中共纳入85例脓毒症心肌病患者,选取时间为2018年1月—2023年10月,进行回顾性分析。将所有患者...目的探讨丹红注射液联合左西孟旦对脓毒症心肌病患者N-末端B型脑钠肽前体(NT-proBNP)、肌钙蛋白I(cTnⅠ)水平及血流动力学的影响。方法研究中共纳入85例脓毒症心肌病患者,选取时间为2018年1月—2023年10月,进行回顾性分析。将所有患者根据治疗方法的不同分为A组(42例,左西孟旦治疗)和B组(43例,丹红注射液联合左西孟旦治疗),14 d为1个疗程,两组患者均进行为期2个疗程的治疗。将两组患者治疗前后NT-proBNP、cTnⅠ水平的变化进行对比,将两组患者治疗前后中医证候积分进行比较,将两组患者治疗前,治疗后24、48、72 h血流动力学指标、心功能指标水平及miR-133a、miR-499a-5p、miR-21-3指标水平进行对比。结果治疗后两组患者NT-proBNP、cTnⅠ指标水平均相较于治疗前下降,且B组患者上述指标比A组下降;治疗后两组患者主症、次症中医证候积分均相较于治疗前下降,且B组患者上述评分比A组低;治疗后不同时间点B组患者心率(HR)均相较于A组下降,随着治疗时间的延长,两组患者HR呈下降趋势,而平均动脉压(MAP)呈上升趋势;治疗后不同时间点两组患者左心室射血分数(LVEF)均相较于治疗前上升,B组患者治疗72 h LVEF指标水平相较于A组上升,治疗后不同时间点两组患者左室舒张末期内径(LVEDD)均相较于治疗前上升,B组患者治疗72 h LVEDD指标水平相较于A组下降(均P<0.05);治疗后两组患者miR-133a、miR-499a-5p、miR-21-3指标水平均相较于治疗前下降,且B组患者上述指标比A组下降(均P<0.05)。结论丹红注射液联合左西孟旦应用于脓毒症心肌病患者的治疗中,可有效缓解患者临床症状,减轻心肌受损程度,降低miRNAs相关指标的表达水平,有助于血流动力学指标的改善。展开更多
基金supported by the National Key Research and Development Program of China(2019YFA0905400)the National Natural Science Foundation of China(32270095)the Open Funding Project(MMLKF23-08)of State Key Laboratory of Microbial Metabolism.
文摘Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools.In this work, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs(PAMs) based on bioinformatics analysis combined with a plasmid interference assay in S. spinosa. By delivering editing plasmids containing a designed mini CRISPR array(repeat+self-targeting spacer+repeat) and repair templates, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the restriction-modification(RM) systems was systematically disarmed. We showed that by disarming one type I RM system(encoded by A8926_1903/1904/1905) and two type Ⅱ RM systems(encoded by A8926_1725/1726 and A8926_2652/2653) simultaneously, the transformation efficiency of the replicative and integrative plasmids(pSP01 and pSI01) was increased by approximately 3.9-fold and 4.2-fold, respectively. Using the engineered strain with simultaneous knock-out of these three RM genes as the starting strain, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster(BGC) as well as gene insertion at high efficiency. Collectively, we developed a reliable and highly efficient genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.
文摘目的探讨丹红注射液联合左西孟旦对脓毒症心肌病患者N-末端B型脑钠肽前体(NT-proBNP)、肌钙蛋白I(cTnⅠ)水平及血流动力学的影响。方法研究中共纳入85例脓毒症心肌病患者,选取时间为2018年1月—2023年10月,进行回顾性分析。将所有患者根据治疗方法的不同分为A组(42例,左西孟旦治疗)和B组(43例,丹红注射液联合左西孟旦治疗),14 d为1个疗程,两组患者均进行为期2个疗程的治疗。将两组患者治疗前后NT-proBNP、cTnⅠ水平的变化进行对比,将两组患者治疗前后中医证候积分进行比较,将两组患者治疗前,治疗后24、48、72 h血流动力学指标、心功能指标水平及miR-133a、miR-499a-5p、miR-21-3指标水平进行对比。结果治疗后两组患者NT-proBNP、cTnⅠ指标水平均相较于治疗前下降,且B组患者上述指标比A组下降;治疗后两组患者主症、次症中医证候积分均相较于治疗前下降,且B组患者上述评分比A组低;治疗后不同时间点B组患者心率(HR)均相较于A组下降,随着治疗时间的延长,两组患者HR呈下降趋势,而平均动脉压(MAP)呈上升趋势;治疗后不同时间点两组患者左心室射血分数(LVEF)均相较于治疗前上升,B组患者治疗72 h LVEF指标水平相较于A组上升,治疗后不同时间点两组患者左室舒张末期内径(LVEDD)均相较于治疗前上升,B组患者治疗72 h LVEDD指标水平相较于A组下降(均P<0.05);治疗后两组患者miR-133a、miR-499a-5p、miR-21-3指标水平均相较于治疗前下降,且B组患者上述指标比A组下降(均P<0.05)。结论丹红注射液联合左西孟旦应用于脓毒症心肌病患者的治疗中,可有效缓解患者临床症状,减轻心肌受损程度,降低miRNAs相关指标的表达水平,有助于血流动力学指标的改善。
