Fig. 5: dxCas9(3.7) expands the range of targetable scRNA target sites by recognizing alternative PAMs. | Nature Communications

Fig. 5: dxCas9(3.7) expands the range of targetable scRNA target sites by recognizing alternative PAMs.

From: Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements

Fig. 5

a CRISPRa with dxCas9(3.7) displayed activity on non-NGG PAM sites with AGA, AGC, AGT, CGA, CGC, CGT, GGA, GGC, GGT, TGA, TGC, TGT, GAA, GAT, CAA sequences. CRISPRa activity with dxCas9(3.7) on non-NGG PAM sites was generally lower (6-fold to 89-fold activation relative to a control without a scRNA) compared to the AGG PAM site (188-fold activation). Sp-dCas9 also displayed moderate CRISPRa activity at non-NGG PAM sites with AGA, CGA, GGA, GGC, GGT, TGA sequences, consistent with published reports6. Reporter plasmids were constructed by replacing the AGG PAM site for the J306 target in the J3-J23117-mRFP1 reporter with alternative PAM sequences that have been previously reported to be recognized by dxCas9(3.7) in human cells6. The (−) sign indicates a control expressing the original reporter with the AGG PAM and the CRISPRa components with Sp-dCas9, the activation domain and no scRNA. b dxCas9(3.7) can activate promoters that cannot be activated by Sp-dCas9. When the scRNA target at the optimal position (−81 to the TSS) has an AGG PAM site, both Sp-dCas9 and dxCas9(3.7) increased gene expression by 50-fold. When the scRNA target at the optimal position has an AGT PAM site, only dxCas9(3.7) displayed a sevenfold increase in gene expression while Sp-dCas9 was inactive. The reporter gene has a target with an AGG PAM (M1) and a target with an AGT PAM (M2) upstream of a BBa_J23117 minimal promoter. In reporter gene A, the AGG target was located −81 to the TSS on the non-template strand and the AGT target was located −76 to the TSS on the non-template strand. In reporter gene B, 5 bases were inserted upstream of the −35 region, shifting the locations of the AGG target and AGT target to −86 and −81, respectively. The (−) sign indicates a negative control strain that contains the reporter plasmid and a plasmid expressing Sp-dCas9, the activation domain and an off-target scRNA (J206). Bars in a and b represent the averagerom the E. coli promoter±standard deviation calculated from n = 3 biologically independent samples. Source data are provided as a Source Data file.

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