a, Genomic context of the CRISPR system in the genetic host (S. islandicus RJW007) and in mutant strains. A1 and A2 denote two different CRISPR arrays, the orientations of which are indicated with arrows. b, PCR verification of Δtype I-A mutants. A representative Sulfolobus transformant with integrated type I-A knockout plasmid was grown in dextrin-tryptone liquid medium, and the cell cultures were plated on dextrin-tryptone plates containing 5-fluoroorotic acid (5-FOA, 50 µg mg−1), uracil (20 µg ml−1), and agamatine (1 mg ml−1). Seven randomly selected 5-FOA-resistant (5-FOAR) colonies were screened using the primers that bind outside of the flanking homologous regions to confirm the type I-A deletion. A representative Δtype I-A mutant was further colony purified for subsequent experiments. The expected sizes of the PCR products amplified from the genomic DNA of the parental strain (referred to wild type, wt) and the Δtype I-A mutant are 8,830 base pairs (bp) and 3,001 bp, respectively. The minus symbol denotes a negative control (using water as the template for PCR). L, log-2 DNA ladder (NEB). Seven biological replicates were screened. c, PCR analysis of the RJW007 Δtype I-AΔcsx1 mutant and its parental strain RJW007 Δtype I-A using primers that anneal to the outside of the flanking homologous regions of csx1, generating amplicons of 2,312 bp and 3,650 bp, respectively. Minus symbol, negative control (using water as the template for PCR). L, Gene Ruler Express DNA ladder (Thermo Scientific). The experiment carried out once. d, Plaque counts for the three strains tested (n = 3 biological replicates).