Fig. 1: The specificity of CRISPR arrays among different CRISPR-Cas systems and the differences in defense activity of different CRISPR-Cas systems.

a CRISPR-Cas loci distributed on HB27 megaplasmid pTT27 and chromosome. CRISPR arrays are numbered and categorized into 3 different types based on their Repeat variants, indicated by different colors. And the direction of their transcription is marked with arrows. b Sequences and predicated secondary structures of 3 different types of HB27 CRISPR repeats. Repeat-1 and Repeat-3 exhibit similarities in structure and sequence, with the differing bases between them highlighted in red. c Schematic diagram for the assessment of the interference activity of endogenous CRISPR-Cas systems through the transformation of exogenous plasmids. (1), (2) and (3) represent three plasmid interference strategies respectively: (1) and (2) use an interference plasmid which carry an artificial mini-CRISPR array to produce crRNAs to target the genome or the plasmid itself, (3) involves an invader plasmid carrying a protospacer designed based on the spacer sequence in endogenous CRISPR arrays. d, e, f Interference plasmids (crRNA-DNApolyα) built on three repeats and different spacers targeting the DNApolyα gene of T. thermophilus HB27 were used to detect the interference activity in a series of CRISPR-Cas system deletion strains and wild-type strain (WT). The combination diagram of repeats and spacers is shown in rectangles and diamonds of different colors. Non-target plasmid (crRNA-DNApolyα-NT) was used as control. g Results of plasmid transformation of T. thermophilus HB27 with self-targeting mini-CRISPR plasmids or invader plasmids containing protospacers for different endogenous CRISPR-Cas systems. gyrA, hoxN, and puc represent the three target genes located on chromosome, megaplasmid, and exogenous plasmid, respectively. Spacers in the mini-CRISPR plasmid were designed based on the sequences of these three genes. Protospacer C2S1/C4S1/C6S1 represents the invader plasmids carrying a target sequence of spacer 1 in CRISPR arrays 2, 4, and 6, which were used to detect the interference activity of III-AB, I-C, and I-B CRISPR-Cas systems, respectively. Non-target plasmid was used as control. To better calculate the number of colonies, the volume of bacterial suspension spread on the plates for the I-B group is only half of that for the I-C group. h The comparison of the copy number of T. thermophilus HB27 genome with the exogenous plasmid through quantitative PCR. The copy number of HB27 genome was normalized to 1, and the relative copy number of plasmid was determined accordingly. The data show means of three biological replicates. Error bars indicate the standard deviations (SD). Between-group significance was determined using the unpaired two-sample t-test (*P < 0.05, **P < 0.01, and ***P < 0.001). And ns indicates no significance.