Development of an inducible Cas9 nickase and PAM-free Cas12a platform for bacterial diagnostics

Hu, Yuanzhao; Qiao, Yuefeng; Li, Xiuqing; Xiang, Zhenbo; Wan, Yi; Wang, Peng; Yang, Zhiqing

doi:10.1016/j.talanta.2023.124931

Cited by 6 publications

(1 citation statement)

References 26 publications

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“…While there are many efforts to loosen specific PAM requirements 16,[24][25][26][27][28][29] and an ongoing search to identify [30][31][32][33][34][35] or engineer 16,[36][37][38][39][40] PAM-less variants, most lack simplicity, require multiple steps for amplification, and aren't versatile enough to be used with other Cas enzymes. Hence, it is very desirable to develop an easier method with fewer steps and fewer constraints.…”

Section: Introductionmentioning

confidence: 99%

PAM-free diagnostics with diverse type V CRISPR-Cas systems

Rananaware,

Meister,

Shoemaker

et al. 2024

Preprint

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Type V CRISPR-Cas effectors have revolutionized molecular diagnostics by facilitating the detection of nucleic acid biomarkers. However, their dependence on the presence of protospacer adjacent motif (PAM) sites on the target double-stranded DNA (dsDNA) greatly limits their flexibility as diagnostic tools. Here we present a novel method named PICNIC that solves the PAM problem for CRISPR-based diagnostics with just a simple ~10-min modification to contemporary CRISPR-detection protocols. Our method involves the separation of dsDNA into individual single-stranded DNA (ssDNA) strands through a high-temperature and high-pH treatment. We then detect the released ssDNA strands with diverse Cas12 enzymes in a PAM-free manner. We show the utility of PICNIC by successfully applying it for PAM-free detection with three different subtypes of the Cas12 family - Cas12a, Cas12b, and Cas12i. Notably, by combining PICNIC with a truncated 15-nucleotide spacer containing crRNA, we demonstrate PAM-independent detection of clinically important single-nucleotide polymorphisms (SNPs) with CRISPR. We apply this approach to detect the presence of a drug-resistant variant of HIV-1, specifically the K103N mutant, that lacks a PAM site in the vicinity of the mutation. Additionally, we successfully translate our approach to clinical samples by detecting and genotyping HCV-1a and HCV-1b variants with 100% specificity at a PAM-less site within the HCV genome. In summary, PICNIC is a simple yet groundbreaking method that enhances the flexibility and precision of CRISPR-Cas12-based diagnostics by eliminating the restriction of the PAM sequence.

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