HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation

Abstract

Rapid molecular diagnostic technology is very useful in many areas, including public health, environmental testing and criminal investigation. We recently showed that Cas12a had trans-cleavage activity upon collateral single-stranded DNA (ssDNA), with which the HOLMES platform (one-HOur Low-cost Multipurpose highly Efficient System) was developed. Here, we combine the thermophilic Cas12b, which also has the ssDNA trans-cleavage activity, with Loop-5 Mediated Isothermal Amplification (LAMP), and create HOLMESv2. In HOLMESv2, LAMP amplification and Cas12b trans-cleavage can be integrated into a one-step system with a constant temperature, which therefore brings much convenience in nucleic acid detection. Moreover, we also simplify the RNA detection procedures in HOLMESv2, using an RNA-dependent DNA polymerase for amplification and therefore omitting an extra reverse transcription step. 10

Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics

“…However, we observed more than background fluorescence signals in several point-mutated sequences, suggesting that detection systems based on Cas12a may partially tolerate point mutation at certain positions. These data are consistent with a previous study, indicating that Cas12b-based nucleic acid detection platform partially tolerated point mutation 30 . We also detected the fluorescence signal of several 1-nt deletion or insertion sequences, and these sequences showed little fluorescence signal ( Supplementary Fig.…”

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

“…Compared to previously reported CRISPR-based nucleic acid detection, 14,16,18,23,24,25 our versatile and robust AIOD-CRISPR has some distinctive advantages. First, AIOD-CRISPR system is a true single reaction system.…”

“…11,12 For example, some Cas nucleases (e.g., Cas12a, Cas12b and Cas13a) perform strong collateral cleavage activities in which a crRNA-targetbinding activated Cas can indiscriminately cleave surrounding non-target single-stranded nucleic acids. 13,14,15,16,17 By combining with RPA preamplification, Cas13 and Cas12a have, respectively, been used to develop SHERLOCK (Specific High-sensitivity Enzymatic Reporter UnLOCKing) system 18 and DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) system 14 for highly sensitive nucleic acid detection. Apart from RPA method, some CRISPR-Cas-based nucleic acid sensors utilized the LAMP and PCR approaches, for instance, the CRISPR-Cas12b-assisted HOLMESv2 platform.…”

“…Apart from RPA method, some CRISPR-Cas-based nucleic acid sensors utilized the LAMP and PCR approaches, for instance, the CRISPR-Cas12b-assisted HOLMESv2 platform. 16 However, these CRISPR-Cas-based detection methods typically require separate nucleic acid pre-amplification and multiple manual operations, which undoubtedly complicates the procedures and brings about contaminations.…”

Rapid, field-deployable nucleobase detection and identification using FnCas9

“…The rise of CRISPR Cas9 based approaches for biosensing nucleic acids has opened up a broad diagnostic portfolio for CRISPR products beyond their standard genome editing abilities 1,2 . In recent times, CRISPR components have been successfully used for detecting a wide variety of nucleic acid targets such as those obtained from pathogenic microorganisms or disease-causing mutations from various biological specimens [3][4][5][6][7][8][9][10] . At the heart of such a detection procedure lies the property of CRISPR proteins to accurately bind to target DNA or RNA, undergo conformational changes leading to cleavage of targets generating a reporter-based signal outcome [11][12][13][14][15] .…”