Figure 3.

Cas12a does not trans-cleave invasive ssDNA in bacteria. (A) Right: Schematic for plasmid co-conjugation and cleavage in P. aeruginosa PAO1 strain. Two plasmids were conjugated simultaneously from different donors into the same recipient cell. LbCas12a was co-expressed in the recipient cell with crRNA complementary to pTarget but not pTrans. A parallel co-conjugation mating using pTrans and pTarget(-PS) (identical to pTarget but lacking the protospacer and PAM) was carried out as a control. Conjugation efficiency was assessed using antibiotic selection specific to each plasmid and represented as the transconjugant to donor ratio (T/D). Data are presented as mean ± SEM (n = 3); **P= 0.0036 by unpaired, two-sided Student's t test; ns, not significant (P> 0.05) (B) Right: Schematic for phage co-infection in E. coli. Bacterial lawns of E. coli BW25113 F’ strain co-expressing LbCas12a and λ-targeting or non-targeting crRNA were singly infected or co-infected with ssDNA phage M13 and dsDNA phage λvir. Strains were infected with either low titer (low), high titer (high), or ten-fold serial dilutions (serial) of phage as indicated.