Recent advances in CRISPR-based single-nucleotide fidelity diagnostics

Abstract

Accurate point-of-care (PoC) detection of single nucleotide variants (SNVs) can support rapid and cost-effective clinical decision-making in tasks such as diagnosing pathogenic genetic variants, identifying pathogen resistance, or tracing viral lineage differentiation. Traditional nucleic acid diagnostics involving PCR and sequencing lack PoC applicability. CRISPR-based diagnostics (CRISPRdx) offer the necessary operational simplicity and ability to integrate specific nucleic acid sequence detection with isothermal amplification. However, achieving single-nucleotide fidelity is not self-evident and often requires empirical optimization. This Review explores recent strategics aimed at refining CRISPRdx specificity for SNV detection including various ways of tactical guide RNA (gRNA) design, fine-tuned effector selection, and improved reaction conditions. While the approaches described here are functional and can be occasionally combined, they often require optimizations to support specific clinical aims. Looking ahead, leveraging computational and AI tools for gRNA design, and harnessing newly discovered CRISPR systems, will broaden applicability and improve precision detection of CRISPRdx in diverse clinical settings.

Fig. 1. Schematic overview of trans cleavage-based CRISPRdx using a fluorescent reporter.

Cas12 or Cas13 can pair with a gRNA to form a ribonucleoprotein complex that recognizes a target sequence. Most Cas12 and some Cas13 proteins respectively require a protospacer adjacent motif (PAM) or protospacer flanking sequence (PFS). The complex initiates cis-cleavage of the matched target and proceeds to trans-cleave single stranded nucleic acids without specific sequence requirements. Cas12 and Cas13 can trans-cleave ssDNA and ssRNA reporters respectively, which can be visualized using a quenched fluorophore reporter.

Fig. 2

Schematic overview of strategies for optimizing single-nucleotide CRISPRdx.

Fig. 3. Schematic overview of gRNA design approaches to improve single-nucleotide specificity.

Most Cas12 and Cas13 gRNAs consist of a spacer and a direct repeat (Cas9 requires an additional trans-activating crRNA [tracrRNA]). For some Cas proteins, the spacer contains a defined window, referred to as the seed region, in which mismatches are poorly tolerated. gRNAs can be designed strategically by making use of SNV-dependent PAM (de)generation. Spacer engineering for improved specificity can involve introduction of synthetic mismatches, truncation or chemical modification. Applied machine learning models can be adopted for generation of high-fitness gRNAs with low off-target predictions.

Fig. 4. Overview of CRISPR classification and diversification.

Protein engineering of CRISPR orthologs can result in Cas proteins (such as AsCas12a Ultra), with altered activity, specificity or PAM preference. This overview does not include the newly proposed Class 1 Type VII system, with Cas14-based RNase activity^,. The proposed Cas14 in these newly identified systems is different from the previously published Type V-F Cas14 proteins, which we refer to as Cas12f throughout this Review. Exceptionally, although classified as a Type V effector, Cas12a2 was shown to detect RNA in cis, and collaterally cleave ssRNA, ssDNA and dsDNA in trans^,.

Fig. 5. Schematic overview of reaction conditions that impact CRISPRdx specificity.

Divalent metals, salts and buffering agents, as well as small molecule additives can be titrated to enhance single-nucleotide specificity. Competitive nucleic acids can be used as additives to disfavor mismatches, yielding higher specificity. Stoichiometry between different components in the CRISPRdx reaction can also reduce off-target activity (graphic shows stoichiometry between Cas protein and gRNA as an example).