Extended Data Fig. 4: Design of CRISPRi-based circuit to respond to SNP base identity.

(a) Schematic of the strain used in Fig. 3h,i (B. subtilis SEN2^#1-# or B. subtilis SEN2^#2-#, where the superscript # indicates the SNP base pair the sgRNA is designed to target, and the -# indicates the SNP base pair on the genome). The SEN2^ΔDT construct containing the SNP is inserted at the amyE locus on the genome and a cassette encoding constitutive expression of dCas9 and a single sgRNA is inserted at the sacA locus. (b) Three sgRNA’s targetting the sense strand, each containing a sequence corresponding to a different SNP base. B. subtilis SEN2^ΔDT encoding a T at the SNP position was engineered to consitutively express dCas9 and one of these three sgRNA’s targeted to the SNP region, yielding the strains B. subtilis SEN2^T1-T, B. subtilis SEN2^G1-T, and B. subtilis SEN2^A1-T. GFP expression in strains B. subtilis SEN2^ΔDT (terminator excised), B. subtilis SEN2 (terminator present), B. subtilis SEN2^T1-T, B. subtilis SEN2^G1-T, and B. subtilis SEN2^A1-T are plotted. Targetting the sense strand leads to high GFP repression (3-fold greater than DT42), irrespective of whether the sgRNA is a perfect match (B. subtilis SEN2^T1-T) or contains a single mismatch (B. subtilis SEN2^G1-T, B. subtilis SEN2^A1-T) with the target sequence. (c) Three sgRNA’s targetting the antisense strand, each corresponding to a different base at the SNP location. dCas9 and these sgRNA’s were constitutively expressed in B. subtilis SEN2^ΔDT encoding a T at the SNP position, yielding B. subtilis SEN2^T2-T, B. subtilis SEN2^A2-T, and B. subtilis SEN^C2-T. GFP expression is shown in the bar plot. When the sgRNA matched the target sequence (B. subtilis SEN2^T2-T), GFP expression was repressed, but not when there was a single mismatch (B. subtilis SEN2^A2-T, B. subtilis SEN2^C2-T). Experimental details are provided in the Methods. The bars show the average of three replicates (dots) performed on different days.