Journal: Applied and Environmental Microbiology

Article Title: Detection and Quantification of Functional Genes of Cellulose- Degrading, Fermentative, and Sulfate-Reducing Bacteria and Methanogenic Archaea

doi: 10.1128/AEM.01285-09

Figure Lengend Snippet: Sequences used for the design of the cel5 , cel48 , hydA , dsrA , and mcrA primer sets

Article Snippet: Pure cultures of the following microorganisms were grown in the laboratory, and their genomic DNAs were used as positive or negative controls for PCR: Escherichia coli , Clostridium cellulovorans (ATCC 35296), Ruminococcus flavefaciens (ATCC 49949), and Fibrobacter succinogenes strain B1 (ATCC 51214).

Techniques:

Journal: bioRxiv

Article Title: Polyvalent Guide RNAs for CRISPR Antivirals

doi: 10.1101/2021.02.25.430352

Figure Lengend Snippet: pgRNAs can robustly suppress viral spread in higher organisms ( Nicotiana benthamiana ). (A) A pgRNA for RfxCas13d was designed to target two sequences in a tobacco mosaic virus (TMV) variant replicon (TRBO-GFP) genome. The targets had 30% (6 out of 23) sequence divergence. MP: movement protein. GFP: green fluorescent protein. (B) Leaves were infiltrated with a suspension of A. tumerfaciens harbouring plasmids for the transient expression of RfxCas13d; a pgRNA, its two “monovalent” counterpart gRNAs, or a non-targeting (NT) gRNA; and replication-competent TRBO-GFP. Representative images of leaves illuminated under UV light three days after infiltration show the extent of viral spread by GFP expression; see Figure S9 for additional leaf images. Viral spread is suppressed by Cas13 RNPs with gRNAs and strongly by Cas13 RNPs with pgRNAs, but not Cas13 RNPs with a non-targeting (NT) gRNA. (C) Quantitative reverse-transcription PCR (qRT-PCR) of leaf RNA after transient expression demonstrates that pgRNAs are capable of more effectively reducing viral RNA levels than their monovalent counterparts in a higher organism (two-sided T-test; * p < 0.05, ** p < 0.01). N = 4 leaves each for gRNA.

Article Snippet: The DNA sequences of the plant codon optimized Cas13d-EGFP with the Cas13d from Ruminococcus flavefaciens (RfxCas13d) flanked by two nuclear localization signal (NLS) was amplified from plasmid pXR001 (Addgene #109049) using Q5 high fidelity of DNA polymerase (NEB).

Techniques: Variant Assay, Sequencing, Expressing, Quantitative RT-PCR