CRISPR‐based biosensors often rely on colorimetric, fluorescent, or electrochemical signaling mechanism, which involves expensive reporters and/or sophisticated equipment. Here, we demonstrated a simple, inexpensive, nonoptical, and sensitive CRISPR‐Cas12a‐based sensing platform to detect ssDNA targets by sizing double‐stranded λ DNA as novel report molecules. In this platform, the size reduction of λ DNA was quantified by gel electrophoresis analysis. We hypothesize that the massive
The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.
more » « less- Award ID(s):
- 2144823
- NSF-PAR ID:
- 10369269
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 14
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. 8377-8391
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract CRISPR‐based biosensors often rely on colorimetric, fluorescent, or electrochemical signaling mechanism, which involves expensive reporters and/or sophisticated equipment. Here, we demonstrated a simple, inexpensive, nonoptical, and sensitive CRISPR‐Cas12a‐based sensing platform to detect ssDNA targets by sizing double‐stranded λ DNA as novel report molecules. In this platform, the size reduction of λ DNA was quantified by gel electrophoresis analysis. We hypothesize that the massive
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Cas12a is an RNA‐guided DNA endonuclease of the type V‐A CRISPR‐Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in
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Abstract CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.
