Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level

Abstract

DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays. We further leveraged the microdroplet-enabled confinement effect to perform an ultralocalized droplet Cas12a assay, obtaining excellent specificity and single-molecule sensitivity. Moreover, we demonstrated its versatility and quantification capability by direct counting of diverse virus’s DNAs (African swine fever virus, Epstein–Barr virus, and Hepatitis B virus) from clinical serum samples with a wide range of viral titers. Given the flexible programmability of crRNA, we envision this amplification-free technique as a versatile and quantitative platform for molecular diagnosis.

Definition of CRISPR Cas12a Trans-Cleavage Units to Facilitate CRISPR Diagnostics

“…Non-paired ssDNA FQ-reporters are the substrates for Cas12a trans-cleavage reactions, and the sequences may affect the cleavage efficiencies (Li et al, 2018a). We first tested four homopolymers of 6 nucleotides in length, including poly-adenine (A), poly-cytosine (C), poly-guanine (G), and poly-thymine (T), respectively, and found Cas12a trans-cleaved the poly-C reporter with the highest efficiency but failed to cleave the poly-G reporter (Figures 1A,B), which was consistent with the previous findings (Yue et al, 2021). Along with the elongation of the ssDNA reporter sequence, the v g gradually increased and reached the peak when the sequence was longer than 8 nucleotides (Figures 1C,D).…”

CRISPR-Cas12a-Based Detection for the Major SARS-CoV-2 Variants of Concern

“…To improve the detection limit, Yue et al described a droplet Cas amplification-free assay in picoliter-sized droplets to increase local molecule concentration and enhance reaction efficiency ( 54 ). They demonstrated a low limit of 17.5 copies/μl virus DNA in their system ( 55 ). Shi et al described CRISPR-Cas12a only amplification network (CONAN) as a novel target amplification free detection with attomolar sensitivity of genomic DNA ( 42 ).…”

Recent Improvements in CRISPR-Based Amplification-Free Pathogen Detection

“…In comparison with the bulk reaction conditions in microliter volume, the created picoliter-sized Cas13a system demonstrates more than 10,000 times enhancement in detection sensitivity and enables absolute target RNA quantitation ( Tian et al, 2021 ). Using the same strategy, the Cas12a-based amplification-free system is then developed to perform an ultralocalized droplet assay, obtaining single-molecule sensitivity for quantitative target DNA detection ( Yue et al, 2021 ). In a similar way, another group develops a platform called CRISPR-based amplification-free digital RNA detection (SATORI) through combining Cas13 detection and microchamber-array technologies, enabling the detection of target RNA at concentrations as low as 5 fM in 5min ( Shinoda et al, 2021 ).…”

“…Taken together, the present amplification-free methods may use different diagnostic principles and have varied LOD and operational convenience. Albeit some amplification-free CRISPR-Dx methods have demonstrated high detection sensitivity within an acceptable detection time ( Hajian et al, 2019 ; Zhou et al, 2020 ; Balderston et al, 2021 ; Sheng et al, 2021 ; Tian et al, 2021 ; Yue et al, 2021 ), the requirement of special chips and instruments as well as the detection cost will limit their wide application and need to be further solved.…”