PAMless CRISPR Detection via Engineered Cas12a
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Solution Overview
Problem
Current CRISPR-based detection methods require a PAM sequence for efficient targeting and detection of nucleic acids, limiting their application to sequences with nearby PAM sequences. Additionally, these methods often necessitate separate amplification and detection steps, increasing time and reducing sensitivity.
Innovation Solution
The development of PAMless CRISPR-based detection systems, such as PICNIC, which utilize Cas12a enzymes to detect target polynucleotides without the need for a PAM sequence. This is achieved through a single-pot reaction that combines isothermal amplification and CRISPR/Cas detection at elevated temperatures, allowing for rapid and sensitive detection of nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional CRISPR-based detection methods are used, then detection specificity is improved, but application scope is limited due to PAM sequence requirement
Solution Approach 1:
The invention changes the biochemical parameters of the CRISPR system by using engineered Cas12a variants with altered PAM specificities. These engineered enzymes can recognize different PAM sequences (such as TTT, TTA, TCT) in addition to the canonical T rich PAM sequences, thereby expanding the range of targetable nucleic acid sequences while maintaining detection specificity through guided binding.
2Measurement precision
If separate amplification and detection steps are used, then detection sensitivity is improved, but assay time is increased
Solution Approach 1:
The invention merges the pre-amplification and CRISPR detection steps into a single-pot reaction. The engineered Cas12a enzymes maintain stability and activity at the elevated temperatures (60-65°C) used for isothermal amplification, allowing both amplification and detection to occur simultaneously in the same reaction vessel, thereby reducing assay time while preserving detection sensitivity.
Solution Approach 2:
The engineered Cas12a enzymes possess multi-functionality by being able to operate in two distinct roles: (1) as amplification enzymes that function at elevated temperatures during the pre-amplification step, and (2) as detection enzymes that perform specific target recognition and trans-cleavage during the CRISPR detection step. This dual functionality enables the single-pot reaction design.
3Productivity
If Cas enzymes are operated at elevated temperatures, then single-pot reaction is enabled, but enzyme stability is reduced
Solution Approach 1:
The invention changes the thermal stability parameter of the Cas12a enzyme through protein engineering. The engineered variants have been optimized to maintain structural stability and catalytic activity at elevated temperatures (60-65°C) that are incompatible with wild-type Cas enzymes, thereby enabling single-pot reactions without sacrificing enzyme stability.
4Measurement precision
If PAM sequence is required for targeting, then detection accuracy is improved, but target coverage is limited
Solution Approach 1:
The invention changes the PAM recognition parameter of the CRISPR system by engineering Cas12a variants with modified PAM specificities. These engineered enzymes can recognize a broader range of PAM sequences including non-canonical sequences (TTT, TTA, TCT), thereby expanding target coverage to include sequences that were previously undetectable while maintaining detection accuracy through guided crRNA binding.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The PICNIC method enables efficient detection of PAMless nucleic acids, including dsDNA and ssDNA, without the need for separate amplification and detection steps. This approach enhances sensitivity and reduces assay time, making it suitable for point-of-care diagnostics.
Implementation Method 1
a Cas12a CRISPR-associated (Cas) enzyme; an sgRNA sequence comprising a CRISPR RNA (crRNA) sequence configured to bind to a single strand of at least one of the one or more target polynucleotides and configured to interact with the Cas12a Cas enzyme to form a CRISPR/Cas complex upon binding of the crRNA sequence to the at least one target polynucleotide
Data Source
AI summary
Described herein are CRISPR/Cas-based methods, systems, compositions, and kits relating to the detection of one or more target polynucleotides that do not require the presence of a PAM sequence. In certain aspects, methods, systems, compositions, and kits utilize Cas12a, customized guide RNA, and an isothermal amplification buffer for polynucleotide detection. In certain aspects, methods, systems, compositions, and kits as described herein can detect SARSCoV-2, and variants thereof; hepatitis C virus (HCV), and variants thereof, human immunodeficiency virus (HIV) and variants thereof, among others. In certain aspects, methods, systems, compositions, and kits can be employed in a point-of-care setting without the use of a thermocycler.