非水虻源微生物与武汉亮斑水虻幼虫联合转化鸡粪的研究

【目的】益生微生物在协助亮斑扁角水虻幼虫转化有机废弃物、提高其转化效率方面具有重要的作用,本研究针对非水虻源微生物,开展与水虻联合转化鸡粪的研究,以阐明外源微生物在水虻转化畜禽粪便中的作用,对其转化机制的研究及产业化生产具有重要意义。【方法】采用稀释涂平板的方法进行鸡粪堆肥和猪粪堆肥中细菌的分析,并将筛选到的细菌分别接种到无菌的鸡粪基质中与武汉亮斑水虻幼虫联合转化,通过称重法测定转化后武汉亮斑水虻及鸡粪的重量,评价转化效果及对幼虫的影响,然后将促进转化效果明显的菌株按不同比例进行复配,与武汉亮斑水虻幼虫联合转化新鲜鸡粪,分析复配菌剂对武汉亮斑水虻幼虫转化鸡粪的影响。【结果】结果显示R-07、R-09、F-03和F-06在促进武汉亮斑水虻幼虫生长和鸡粪转化的效果上最为显著。与对照组相比,水虻幼虫转化率分别提高了27.21%、15.00%、9.93%和16.29%;基质减少率分别提高了17.94%、10.42%、7.84%和9.27%。将这4株细菌配制复配菌剂与武汉亮斑水虻幼虫联合转化鸡粪,结果显示复配比例为R-07:R-09:F-03:F-06=4:1:1:1时效果最好,与空白对照相比,武汉亮斑水虻幼虫存活率提高了10.25%,幼虫虫重增加了28.41%,幼虫转化率增加了30.46%,鸡粪减少率增加了7.69%。【结论】添加通过筛选优化的非水虻来源的微生物复合菌剂能够促进水虻高效转化鸡粪,研究结果有助于改善现有的武汉亮斑水虻幼虫转化体系,为开发新型的联合转化工艺、更加有效地处理畜禽粪便奠定基础。     

Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3′ end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3′ GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.     

Rapid and Precise Engineering of the Caenorhabditis elegans Genome with Lethal Mutation Co-Conversion and Inactivation of NHEJ Repair

As in other organisms, CRISPR/Cas9 methods provide a powerful approach for genome editing in the nematode Caenorhabditis elegans. Oligonucleotides are excellent repair templates for introducing substitutions and short insertions, as they are cost effective, require no cloning, and appear in other organisms to target changes by homologous recombination at DNA double-strand breaks (DSBs). Here, I describe a methodology in C. elegans to efficiently knock in epitope tags in 8–9 days, using a temperature-sensitive lethal mutation in the pha-1 gene as a co-conversion marker. I demonstrate that 60mer oligos with 29 bp of homology drive efficient knock-in of point mutations, and that disabling nonhomologous end joining by RNAi inactivation of the cku-80 gene significantly improves knock-in efficiency. Homology arms of 35–80 bp are sufficient for efficient editing and DSBs up to 54 bp away from the insertion site produced knock-ins. These findings will likely be applicable for a range of genome editing approaches in C. elegans, which will improve editing efficiency and minimize screening efforts.