In Vitro CRISPR-Cas12a-Based Detection of Cancer-Associated TP53 Hotspot Mutations Beyond the crRNA Seed Region

Abstract

Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis , exhibits indiscriminate ssDNase activity in trans , which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called “hotspots.” In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.

New design strategies for ultra-specific CRISPR-Cas13a-based RNA detection with single-nucleotide mismatch sensitivity

“…Moreover, we also noticed that the mismatched base pair type and the orientation (in the spacer vs. the target RNA) changed the extent of nuclease activation. Similar observations have been made where non-canonical base pairs can elicit robust Cas9 or Cas12 activation ( 21 , 80 ), and recent work with Cas13 demonstrated that G-U mismatches are the most tolerated ( 33 , 81 ). Future functional and structural studies of Cas13 enzymes will shed light on non-canonical base pairing tolerance, which, in turn, will further guide optimization efforts to select for base pairs within the target that can yield the highest discrimination power.…”

“…However, harnessing these sensitivities for RNA discrimination is challenging, as high target RNA concentrations and/or longer spacers are able to overcome mismatch-induced cleavage defects. Similar observations pertaining to target concentrations have been made with Cas12a, where it has been observed that PCR cycles for target amplification needed to be controlled to prevent triggering off-target effects ( 21 ).…”

“…One successful strategy has been to introduce one or more additional ‘synthetic mismatches’ to achieve discrimination at a critical position ( 8 , 9 , 20 , 31 ). However, where to place the discriminatory and/or synthetic mismatches is not intuitive, needs to be determined empirically for each crRNA-target set and does not always yield robust discrimination ( 21 , 24 , 32 ). Recent work has exploited machine learning principles to discern nucleotide-preferences, mismatch tolerance and design crRNAs for viral detection that are efficient when testing or discriminating genetic variation between samples (ADAPT) ( 33 ), but these designs still require thorough validation.…”

New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity

“…The same principle of detecting cleaved reporters has been harnessed for other CRISPR and/or RNA-guided nucleases such as CRISPR-Cas12 or prokaryotic Argonaute proteins (16,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30).…”

“…One successful strategy has been to introduce one or more additional 'synthetic mismatches' to achieve discrimination at a critical position (16,17,28,39). However, where to place the discriminatory and/or synthetic mismatches is not intuitive, needs to be determined empirically for each crRNA-target set and does not always yield robust discrimination (29,32,40). Recent work has exploited machine learning principles to discern nucleotide-preferences, mismatch tolerance and design crRNAs for viral detection that are efficient when testing or discriminating genetic variation between samples (ADAPT) (41), but still require thorough validation.…”