Figure 2
CRISPR-Cas9 genome editing vector and retargeting crRNA sequences. (A) Diagrammatic representation of the CRISPR-Cas9 vector developed in this study. The requisite CRISPR-Cas9 components, namely S. pyogenes cas9, under the control of the C. acetobutylicum thiolase promoter (Pthl); an sgRNA component, consisting of a gRNA handle and crRNA (2008A) under the control of C. difficile toxin B promoter (PtcdB), and an editing template, containing upstream and downstream chromosomal regions flanking the deletion target site, cloned between AsiSI and AscI restriction sites. The plasmid backbone consists of the E. coli-clostridia shuttle vector pMTL83151. (B) Positions of 20-nt crRNA retargeting sequences within the region targeted for deletion from Tn5398 labelled A–C, corresponding to the 2008A-C sequences listed in (C), along with on- and off-target scores provided by the Benchling CRISPR guide design tool. The protospacer-associated motif (PAM) for each crRNA sequence is also stated. Similarly, locations (D; not to scale) of the three crRNA retargeting sequences (E) present in the three CRISPR-Cas9 vectors utilised for truncation of 234 bp at the 3′-end of pyrE.
