Figure 2.

Inter-domain interaction in Cas3/I-C influences CRISPR interference. (A) An E. coli IYB5101 strain harboring genes encoding Cascade/I-C (IC-1) was used as a surrogate for in vivo experiments. Cas3/I-C and crRNA were expressed through IPTG inducible vectors having kanamycin and spectinomycin antibiotic resistance markers, respectively. The target sequence was inserted into pUC19 vector (T1), whereas an empty pUC19 vector was used as non-target (NT1). Low transformation efficiency indicates functional CRISPR interference. (B) Transformation efficiencies of the strain IC-1 against the target (T1) and the non-target (NT1) for wild-type and variants of Cas3/I-C are shown. In the presence of Cas3/I-C wild-type (pWT) and co-expressed construct of HD and DExD/H domain (pHD+pHL), there was a significant reduction in transformation efficiency (indicated by a red bar). Interference was rendered ineffective when the HD domain mutant (pD48A), Helicase domain mutant (pQ253A and pD395A), CTD deletion mutant (pWTΔCTD), stand-alone nuclease domain (pHD) and stand-alone helicase domain (pHL) were used. Error bar represents standard deviation measured from three independent trials. The boundary of the domain variants are as follows: HD domain (pHD) 1–248; DExD/H domain (pHL) 249–800; WT without CTD (pWTΔCTD) 1–709. (C) Highly conserved residues in the CTD domain from type I-C system are shown. Red dots indicate those residues from B. halodurans that are chosen for alanine-scanning mutagenesis. (D) Homology model of Cas3/I-C from B. halodurans based on crystal structure of Cas3/I-E [PDB ID: 4QQX] shows the organization of nuclease (HD in cyan), DExD/H helicase (RecA1 in pink and RecA2 in orange) and CTD (in blue) domains. DNA is shown as a rod in green. Amino acids (K742, K743, Q745, Q746 and Y747) from CTD domain that are possibly interacting with target DNA are shown. (E) A 100 bp target DNA harboring functional PAM sequence TTC is depicted. Target sequence represents the strand, which is complementary to crRNA. (F) CTD point mutants were tested for their nuclease activity. Cascade/I-C was incubated with target DNA in the reaction mixture containing Mg²⁺ and ATP to form R-loop. Wild-type or CTD mutant was added to the reaction and the cleavage was monitored. Proteinase K was added to release residual Cascade bound DNA fraction. DNA was analyzed using 20% native PAGE. (G) CTD mutants were tested for their role in CRISPR interference using E. coli IC-1 strain as the surrogate host. Wild-type and CTD mutants were expressed through an IPTG inducible 1R vector (referred to as pWT, pK742A and so on). Transformation efficiency was measured against pUC19 harboring the target sequence with functional PAM TTC (referred to as pT1). Error bar represents standard deviation measured from three independent trials.