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Fig. 4 | BMC Biology

Fig. 4

From: DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system

Fig. 4

Influence of protospacer mutations at the PAM-distal end on DNA interference. a Constructs of altered protospacer sequences. The CC PAM-distal end of protospacer (PS-CC), which has complete complementarity for 32 and 62 nt spacers, was truncated by 6 (PS-T6) or 30 bp (PS-T30) or a mismatch of 6 bp was introduced between the 27th and 32nd positions of the protospacer (PS-M6). b Interference for plasmids with altered protospacer sequences. Plasmids containing either WT or altered target sequence (as shown in a) as well as plasmid without target sequence (PS-NT) were introduced into E. coli cells carrying Cas1-Cas2/3 and WT or +30 Cascade; then, transformation efficiency was estimated (CFU per μg of plasmid DNA). c WT and +30 Cascade binding to DNA with altered protospacer sequences. Binding of WT and +30 Cascade to respective DNA was examined by the EMSA. d Dissociation constants for all substrates. The constants were determined using EMSA. e Dependency of DNA hydrolysis on R-loop length. Cleavage of respective linearized plasmid was monitored in the presence of Cas2/3 and WT or +30 Cascade. Error bars in b and d represent standard deviations for at least three separate experiments (individual data values are provided in Additional file 14)

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