ArticleThe Biochemical journal2026
Target discrimination and PAM profiling of the Thermotoga maritima type I-B CRISPR system.
Article in The Biochemical journal, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
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1 citing paper in PubMed.
- Target discrimination and PAM profiling of the Thermotoga maritima type I-B CRISPR system.The Biochemical journal · 2026Article
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4 authors.
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Abstract
Type I-B CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems represent the most abundant CRISPR subtype in nature and have emerged as powerful tools for endogenous genome editing in diverse prokaryotes. Here we reconstitute and characterize the type I-B1 system from the thermophile Thermotoga maritima (Tma) using purified components. We demonstrate that Tma Cascade requires standalone Cas11 expression, as the cryptic internal translation start site within cas8b1 is non-functional in Escherichia coli. The reconstituted system exhibits canonical type I function including RNA-guided DNA binding, protospacer adjacent motif (PAM)-dependent target discrimination, Cas3-mediated degradation, and seed region interrogation spanning seven PAM-proximal nucleotides. Using next-generation sequencing-based PAM library screens, we define a YYD consensus PAM (Y = C/T; D = G/A/T) with strong discrimination against the array repeat-adjacent sequence (AAC). Comprehensive PAM profiling reveals context-dependent tolerance for non-consensus sequences and identifies numerous intermediate-activity PAMs that may function in priming. Comparison with other characterized type I-B systems reveals a correlation between the Cas8b variant and position -3 specificity, conserved pyrimidine preference at position -2, and variability at position -1. This work establishes a thermostable type I-B platform for biotechnological applications and provides insights into evolutionary mechanisms balancing PAM promiscuity with self-discrimination in the most abundant CRISPR-Cas subtype.
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