CUT-PCR: CRISPR-mediated, ultrasensitive detection of target DNA using PCR

Abstract

Circulating tumor DNA (ctDNA) has emerged as a tumor-specific biomarker for the early detection of various cancers. To date, several techniques have been devised to enrich the extremely small amounts of ctDNA present in plasma, but they are still insufficient for cancer diagnosis, especially at the early stage. Here, we developed a novel method, CUT (CRISPR-mediated, Ultrasensitive detection of Target DNA)-PCR, which uses CRISPR endonucleases to enrich and detect the extremely small amounts of tumor DNA fragments among the much more abundant wild-type DNA fragments by specifically eliminating the wild-type sequences. We computed that by using various orthologonal CRISPR endonucleases such as SpCas9 and FnCpf1, the CUT-PCR method would be applicable to 80% of known cancer-linked substitution mutations registered in the COSMIC database. We further verified that CUT-PCR together with targeted deep sequencing enables detection of a broad range of oncogenes with high sensitivity (<0.01%) and accuracy, which is superior to conventional targeted deep sequencing. In the end, we successfully applied CUT-PCR to detect sequences with oncogenic mutations in the ctDNA of colorectal cancer patients’ blood, suggesting that our technique could be adopted for diagnosing various types of cancer at early stages.

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

“…The chip sits on a hotplate set at 37°C and does not need thermal cycling with temperature control like PCR does. 35,36 Thus, the detection system we developed is much simpler and more compact, ideal for POC applications. 37,38 To extend the detection limit, our system can be integrated with isothermal amplification methods such as recombinase polymerase amplification for sensitive "one pot" target amplification and CRISPR reaction, without the background issues seen in traditional "one-pot" methods.…”

Highly specific enrichment of rare nucleic acids usingThermus thermophilusArgonaute

“…The recently-developed assays Cas9-DASH 10 (Depletion of Abundant Sequences by Hybridization) and CUT-PCR 11 (CRISPR-mediated Ultra-sensitive detection of Target DNA-PCR) take advantage of the low tolerance of CRISPR/Cas proteins to mismatches at the PAM recognition site to discriminate mutant from WT alleles. We examine the discrimination efficiency of CRISPR/Cas9 with a previously reported guide RNA [10][11][12] ( Supplementary Fig. 18a).…”

“…In recent years, researchers have identified various enzymes from archaea and bacteria that can be programmed with nucleic acids to cleave complementary strands, among which CRISPR-Cas have attracted considerable attention as genome-editing tools [1][2][3] . In medical diagnostics, CRISPR-associated nucleases have been used to (1) amplify reporter signals during nucleic acid detection [4][5][6][7][8] , (2) detect unamplified targets immobilized on graphene field-effect transistors 9 , and (3) enrich rare alleles to enhance detection limits of oncogenic sequences [10][11][12] . Despite remarkable progress, the applications of CRISPR-Cas are restricted due, among other things, to their reliance on the protospacer-adjacent motif (PAM), which is absent in many sequences of interest [10][11][12] .…”

Directed evolution studies of a thermophilic Type II-C Cas9

“…Class 1 is defined by multi-subunit CRISPR–Cas effector complexes, whereas Class 2 is characterized by single-subunit effector complexes (Koonin et al, 2017). Due to its simplicity, Class 2 is the easiest to be implemented in gene editing as well as manipulation of cell-free nucleic acids (Barrangou & Doudna, 2016; Gootenberg et al, 2017; Komor, Badran, & Liu, 2017; Koonin & Makarova, 2009; Lee et al, 2017; Wang, La Russa, & Qi, 2016). The Class 2 systems primarily include the Types II (Cas9), V (Cas12), and VI (Cas13) systems where Cas9 and Cas12 are RNA-guided DNA cleavage enzymes and Cas13 is RNA-guided RNA cleavage enzymes, although some DNA cleavage activities have been reported (Gao, Yang, Rajashankar, Huang, & Patel, 2016; Makarova, Zhang, & Koonin, 2017; Smargon et al, 2017).…”