Journal: Communications Biology

Article Title: Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus

doi: 10.1038/s42003-022-03025-4

Figure Lengend Snippet: a Schematic depiction of the in vivo activity assay. Two reporter gene constructs ( luxABCDE and GFP ) as well as expression constructs for RcCas13a variants and crRNA encoded within a CRISPR-locus targeting luxABCDE are introduced into E. coli cells. Upon expression of RcCas13a and crRNA luxA cis -cleavage activity can be monitored by reduction of luminescence, while sequence unspecific trans -cleavage can be assessed by alteration in GFP-fluorescence. b Sequence-specific target cleavage activity ( cis -cleavage) by RcCas13a is followed by bioluminescence upon luciferase expression reduction. Wt RcCas13a and RcCas13aΔCTD reduce bioluminescence in a crRNA luxA dependent manner (red and brown bar), while all HEPN-mutants lost their impact on luciferase expression (blue bars). Scrambling of the sequence in the seed region nt 10–14 of the spacers abrogates reduction of luciferase level by RcCas13a (black bar). c Sequence-unspecific RNA cleavage (bystander-cleavage) was analyzed by measuring GFP-fluorescence upon induction of crRNA luxA and target RNA expression ( luxABCDE ). Wt RcCas13a and RcCas13aΔCTD reduce GFP-fluorescence only upon expression of crRNA luxA and target sequence (red and brown hatched bars). Mutation of any of the catalytic residues in the HEPN-domains leads to an inactive RcCas13a (blue hatched bars). RcCas13a and crRNA expression is induced by addition of arabinose to the growth medium. Luciferase and GFP expression is controlled by anhydrotetracycline and IPTG, respectively. (+): Encoding gene/expression construct is present in the E. coli cells, but was not induced. (−) empty vector control. (H1* = RcCas13a R464A/H469A ; H2* = RcCas13a R464A/H469A ; H1 + 2* = RcCas13a R464A/H469A/R1052A/H1057A ; ΔCTD = Δ C-terminal domain); experiments were performed in three independent replicates ( n = 3).

Article Snippet: The sequences encoding the luciferase subunits luxA, luxB, luxC, luxD and luxE from Photorhabdus luminescens (Uniprot IDs P23146, P19840, P23113, P23148, P19482) were cloned into pASK-IBA5 (IBA Lifesciences), under control of an anhydrotetracycline (aTc) inducible promoter.

Techniques: In Vivo, Activity Assay, Construct, Expressing, CRISPR, Sequencing, Fluorescence, Luciferase, RNA Expression, Mutagenesis, Plasmid Preparation