The CRISPR/Cas12a RNA-guided complexes have a tremendous potential for nucleic acid detection due to its ability to indiscriminately cleave ssDNA once bound to a target DNA. However, the current CRISPR/Cas12a systems are limited to detecting DNA in a picomolar detection limit without an amplification step. Here, we developed a platform with engineered crRNAs and optimized conditions that enabled us to detect DNA, DNA/RNA heteroduplex and methylated DNA with higher sensitivity, achieving a limit of detection of in femtomolar range without any target pre-amplification step. By extending the 3'-or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discovered a new selfcatalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA. We applied this sensitive system to detect as low as 25 fM dsDNA from the PCA3 gene, an overexpressed biomarker in prostate cancer patients, in simulated urine over 6 hours. The same platform was used to detect as low as ~700 fM cDNA from HIV, 290 fM RNA from HCV, and 370 fM cDNA from SARS-CoV-2, all within 30 minutes without a need for target amplification. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs were incorporated in a paper-based lateral flow assay that could detect the target with up to 23-fold higher sensitivity within 40-60 minutes.
MainClass 2 CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPRassociated proteins) systems, such as Cas12a (Cpf1, subtype V-A) and Cas13a (C2c2, subtype VI), are capable of nonspecific cleavage of ssDNA (single-stranded DNA) and RNA, respectively, in addition to successful gene editing. 1-3 This attribute, known as trans-cleavage, is only activated once bound to an activator (ssDNA or dsDNA) that has complementary base-pairing to the guide crRNA. When combined with a FRET-based reporter, a fluorophore connected to a quencher via a short oligonucleotide sequence, the presence of the target activator can be confirmed. This phenomenon has been efficiently harnessed by SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) to reliably detect nucleic acids. 1,[4][5][6][7][8] Research studies have reported that an extended secondary DNA on the guide crRNA for Cas12a or a hairpin RNA structure added to the sgRNA for Cas9 increases the efficiency and specificity of gene editing. 9,10 In addition, chemically modified Cas12a guided-RNA has also been shown to facilitate improved gene correction in mammalian cells through both viral-and non-viral methods compared to the wild-type guide RNA. 11 Though these modifications were employed to utilize the cis-cleavage aspect of the CRISPR/Cas systems, the effects of such alterations on the transcleavage remain unknown.was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.