Journal: Molecular cell
Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28
Figure Lengend Snippet: Heterologous expression of Cas13b mediates knockdown of E. coli essential genes by a double-sided PFS (A) Design of E. coli essential gene screen to determine targeting rules of nucleic acid interference. (B) Manhattan plots of mean spacer depletions mapped over 45 genes and aggregated across normalized gene distance for either the full B. zoohelcum VI-B1 locus (left) or cas13b alone (right), with non-targeting spacers in gray, safely depleted spacers ( > 5σ above mean depletion of non-targeting spacers) above blue line, and strongly depleted spacers (top 1% depleted) above red line. For the full locus, 36,142 targeting spacers and 630 non-targeting spacers passed QC filter. Of the targeting, 367 are strongly depleted and 1672 are safely depleted. For cas13b alone, 35,272 targeting spacers and 633 non-targeting spacers passed QC filter. Of the targeting, 359 are strongly depleted and 6374 are safely depleted. (C) Weblogo of sequence motifs of strongly depleted B. zoohelcum spacers. (D) Normalized PFS score matrix, where each score is the ratio of number of safely depleted B. zoohelcum spacers to total number of spacers for a given PFS, scaled so that maximum PFS score is 1. (E) Spacers targeting kanamycin to validate PFS targeting rules of 5′ PFS (D) and 3′ PFS (NAN or NNA). (F) Schematic of kanamycin validation screen for B. zoohelcum cas13b in E. coli . (G) Results from kanamycin validation screen; spacer abundances versus control for individual B. zoohelcum .
Article Snippet: The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).
Techniques: Expressing, Sequencing