PCR amplification of the leader-proximal CRISPR regions

Sulfolobus islandicus E233S and the cas-deletion strains harboring an empty expression vector or the csa3a-overexpression plasmids were cultured in 10 ml of SCVy medium at 78°C until the OD₆₀₀ reached 0.3. Samples of each culture (0.1 ml) were taken, and total DNA from these cells was used as a PCR template. The leader-proximal regions of two CRISPR loci were amplified by PCR using Taq polymerase, the forward primer CRISPR-F, and the reverse primer CRISPR2S5-R for locus 2; the forward primer CRISPR-F and reverse primer CRISPR1S5-R for locus 1. PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining. PCR products from Δcas6::csa3a and Δcas3Δcmr2αΔcmr2β::csa3a were purified using the Axygen Cleanup Kit, and the expanded bands larger than those of the wt control band (parental band) were excised from the gel and purified using the Axygen DNA Extraction Kit. Purified PCR products were cloned into the T-vector (Takara, Dalian, China), following the manufacturer's instructions. Subsequently, the ligation products were transformed into E. coli DH5α cells. Plasmids from single colonies were purified and sequenced at Qingke (Wuhan, China).