Enhanced selection of efficient targeted genome manipulating agents
Inventors
Peytavi, Regis • Aran, Kiana • GOLDSMITH, BRETT • Kane, Alexander
Assignees
Publication Number
US-11713455-B2
Publication Date
2023-08-01
Expiration Date
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Abstract
For enhanced selection of efficient targeted genome manipulating agents, an apparatus includes first and second chip-based biosensors having one or more sensing surfaces configured to detect biomolecular binding interactions between a nucleic acid sample and a targeted genome manipulating agent functionalized to a capture surface within a sensing range of the one or more sensing surfaces. The first chip-based biosensor uses a nucleic acid sample incubated with a blocking agent that blocks on-target binding and the second chip-based biosensor holds a nucleic acid sample that omits the blocking agent. A measurement apparatus measures first and second sets of response signals produced in response to the biomolecular binding interactions occurring between the nucleic acid sample and the targeted genome manipulating agent. An analysis module determines the genome manipulating efficiency parameters of the targeted genome manipulating agent. A system and a method perform the functions of the apparatus.
Core Innovation
The invention provides an apparatus, system, method, and computer program product for enhanced selection of efficient targeted genome manipulating agents. The apparatus includes a first chip-based biosensor and a second chip-based biosensor individually comprising one or more sensing surfaces on channels of liquid gated field effect transistors made of two-dimensional material, a counter electrode on the chip to incrementally and programmatically adjust a gate voltage of a liquid gate formed over the channels by a sample liquid, and a reference electrode on the chip to detect the voltage of the liquid gate; the first chip-based biosensor receives a first aliquot incubated with a blocking agent selected to bind to a sequence overlapping an on-target sequence and the second chip-based biosensor receives a second aliquot that omits the blocking agent. A measurement controller measures one or more first and second response signals produced in response to the biomolecular binding interactions between the nucleic acid sample aliquots and capture surfaces functionalized with a targeted genome manipulating agent comprising a CRISPR associated protein and a guide RNA, and an analysis module determines genome manipulating efficiency parameters based on comparing the first and second response signals.
The invention addresses limitations in existing screening and validation tools for genome manipulating targeting components by providing comparatively fast and inexpensive electronic biosensing and label-free measuring to improve selection of targeted genome manipulating agents and to enhance off-target putative site discovery. The disclosure contrasts existing in silico, in vitro, and in vivo methods and describes use cases where chip-based field-effect biosensing provides lower cost, faster screening and validation, and the ability to enrich and prepare nucleic acid samples for sequencing after selection of efficient targeted genome manipulating agents.
Claims Coverage
This coverage identifies three independent inventive features from the independent claims.
Apparatus with paired chip-based liquid gated field effect transistor biosensors
First and second chip-based biosensors individually comprising one or more sensing surfaces on channels of liquid gated field effect transistors on a chip, wherein the channels are made of two-dimensional material selected from graphene and molybdenum disulfide; a counter electrode on the chip used to incrementally and programmatically adjust a gate voltage of a liquid gate formed over the channels by a sample liquid; and a reference electrode on the chip used to detect the voltage of the liquid gate.
Differential incubation and comparative measurement for assessing targeted genome manipulating agents
Liquid gated field effect transistors output one or more transistor response signals corresponding to detected biomolecular binding interactions between a nucleic acid sample and capture surfaces functionalized with a targeted genome manipulating agent having a CRISPR associated protein (Cas9, Cas12, Cas13) and a guide RNA, wherein the first chip-based biosensor receives a first aliquot incubated with a blocking agent selected to bind to a sequence overlapping an on-target sequence and the second chip-based biosensor receives a second aliquot that omits the blocking agent; a measurement controller measures one or more first and second response signals and an analysis module determines one or more genome manipulating efficiency parameters based on performing a comparison of the first and second response signals.
Computer-implemented measurement and analysis components
A measurement controller and an analysis module that measure the one or more first and second response signals and determine genome manipulating efficiency parameters, wherein the measurement controller and the analysis module each comprise one or more of hardware circuits, programmable hardware devices, and executable code stored on one or more non-transitory computer readable storage media.
Method for field-effect, label-free detection and efficiency determination
Preparing a first aliquot and a second aliquot where the first aliquot is incubated with a blocking agent configured to bind to a sequence that overlaps an on-target sequence and the second aliquot omits the blocking agent; measuring one or more first and second response signals produced in response to biomolecular binding interactions occurring between the nucleic acid sample aliquots and the targeted genome manipulating agent functionalized to capture surfaces of first and second chip-based biosensors; and determining an efficiency parameter of the targeted genome manipulating agent based on comparatively analyzing the first and second response signals, utilizing field-effect biosensing for label-free detection.
Computer program product for control and comparative analysis
Program instructions on a non-transitory computer readable storage medium executable to control measurement of one or more first and second response signals produced by first and second chip-based biosensors comprising sensing surfaces on channels of liquid gated field effect transistors made of graphene or molybdenum disulfide with counter and reference electrodes, wherein the first biosensor receives a first aliquot incubated with a blocking agent and the second biosensor receives a second aliquot that omits the blocking agent, and to determine one or more genome manipulating efficiency parameters based on performing a comparative analysis of the first and second response signals.
The independent claims collectively define an apparatus, a method, and a computer program product for comparative field-effect, label-free biosensing using paired chip-based liquid gated field effect transistors (blocked versus unblocked aliquots or active versus deactivated Cas pairings) to measure response signals and determine genome manipulating efficiency parameters.
Stated Advantages
Lower costs, faster screening and validation, and better precision for off-target site discovery.
Label-free measuring that is faster, can be manufactured at a lower cost, with higher repeatability and lower complexity than other measurement technologies.
Improves the selection of efficient targeted genome manipulating agents and improves downstream technologies such as amplification, sequencing, and therapeutic uses by allowing such downstream to be more efficiently utilized.
Documented Applications
Enhanced selection of efficient targeted genome manipulating agents.
Screening and validation of genome manipulating targeting components as an alternative or complement to in silico, in vitro, and in vivo methods.
Off-target putative site discovery and enrichment of putative off-target sites for sequencing.
Label-free detection of biomolecular binding interactions using field-effect biosensing (biology gated transistors).
Preparing nucleic acid samples for sequencing after measurement, including fragmenting, adapter ligation, and tagmentation workflows to enable sequencing of captured on-target and off-target regions.
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