High concentration of Cas12a effector tolerates more mismatches on ssDNA

Abstract

Rapid and sensitive pathogen detection cuts off routes of transmission and improves patient outcomes by enabling early treatment and appropriate antibiotic usage. 1 Several rapid methods having different adaptation, cost, sensitivity, and specificity, have been developed for pathogen detection. 2 Rapid pathogen detection, based on detection of nucleic acids via polymerase chain reaction (PCR), is often costly, requires specific equipment, and must be conducted in the laboratory environment. 3 Sequencing technology is still being perfected and cannot be directly used for clinical diagnosis. 4 This highlights the urgent need for a low-cost, simple, and highly

CRISPR-Cas Systems-Based Bacterial Detection: A Scoping Review

“…With regards to the analytical sensitivity of the CRISPR-Cas assays, the limit of detection (LoD) was evaluated in three types of concentration which include copies per mL, CFU per mL and molarity. For studies reporting the LoD in copies per mL, detection of Mycobacterium tuberculosis H37Rv utilizing EnGen LbaCas12a reported the lowest LoD of 1 copy/uL [ 40 ]. Meanwhile, most of the studies reported the LoD in CFU/mL, of which seven studies showed LoD of 1 CFU/mL.…”

PAM-less Exonuclease-assisted Cas12a for visual detection of Vibrio Species

“…Compared to the previous study utilising ssDNA as a cas12a activator without PAM activation [13], the cas12a concentration of the AIOD-CRISPR system is 1.28µM due to buffer incompatibility of cas12a in RPA buffer, while for PECAN it is 0.2µM. Previous research investigating the specificity of cas12a detection of ssDNA template suggested that with higher cas12a enzyme in the reaction, the less specific the cas12a trans-cleavage activity is, as high concentrations of cas12a were shown to decrease the minimum activating bases for crRNA complementary sequence [35]. The high cas12a usage also leads to a detection cost per reaction of $5.4 USD.…”

“…However, it was shown that in ssDNA, a mismatch of the 5' end of crRNA could still lead to cas12a activation, leading to a minimum activation base of 11bp only in GC-rich crRNA, which is 3-bp shorter than using dsDNA. The same research group proposed some strategies; for example, using AT-rich crRNA target strand increases the specificity of ssDNA crRNA detection up to 14-bp minimum activation base [35]. This also suggests that the crRNA ssDNA hybridisation mechanism might be simple, like with the use of RPA fluorophore probe, which generates a free fluorophore per hybridised strand.…”

“…Yet the Addition of 7-nt of DNA might hybridise with target ssDNA strand, and lead to concern to specificity. However, it was shown that with a crRNA extension of 5-nt, the minimum activating bases increased from 11-nt to 16-nt [35], indicating that 3’ extensions of crRNA might not decrease the required bases of activation of cas12a. The use of cas12a could therefore act as a secondary specificity safeguard, together with the RPA assay which is itself highly specific.…”

“…Moreover, a higher concentration of cas12a was reported to lead to decreased specificity of ssDNA template recognition. It was shown that higher concentrations of cas12a are activated by shorter ssDNA sequences, compared to no activation by shorter ssDNA with a lower concentration of cas12a, even with longer incubation time [35]. Hence there might be higher false positive rates of detection in the case when there is a partial crRNA complementary sequence in the RPA amplicon.…”