Methods for measuring and improving CRISPR reagent function
Inventors
Kryukov, Gregory V. • Schlabach, Michael R. • Merkin, Jason J.
Assignees
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Publication Number
US-11078483-B1
Publication Date
2021-08-03
Expiration Date
Abstract
The invention describes a novel system for identifying optimized gRNAs for use in CRISPR/Cas9 genome editing platforms. The invention allows for the determination of specific gene alterations rendered by a particular gRNA, thereby permitting the generation of optimized gRNA libraries.
Core Innovation
The method prepares an optimized guide RNA (gRNA) library by introducing a library of nucleic acid constructs into a population of cells modified to express a Cas9 protein, where each construct comprises a gRNA sequence and a sensor sequence. The sensor sequence comprises the corresponding gRNA target sequence, enabling the sensor sequence to record the outcome of gRNA-directed Cas9 activity. The cells are cultured under conditions permitting expression of the gRNA and the Cas9 protein.
The method amplifies the nucleic acid construct sequences from the cells using polymerase chain reaction (PCR) to obtain amplicons that comprise the gRNA sequence and the sensor sequence. The amplicons are sequenced, and a pattern of alterations in the sensor sequence is determined for each amplicon. A population of gRNAs that produce a desired pattern of alterations in the sensor sequence is then identified.
In certain embodiments, the nucleic acid encoding the Cas9 protein mRNA is introduced into the cell population by electroporation. The document further describes genome-wide or sub-genome-wide gRNA libraries, high-throughput sequencing to determine sensor alteration patterns, and using altered sensor sequence outcomes to select optimized guide RNAs for desired outcomes.
Claims Coverage
The document provides one independent claim covering preparation of an optimized gRNA library using a gRNA-sensor construct readout in Cas9-expressing cells. The claim includes six main inventive features, with an explicit limitation that Cas9 mRNA is introduced by electroporation.
gRNA-sensor constructs in Cas9-expressing cells
Introducing a library of nucleic acid constructs into a population of cells modified to express a Cas9 protein, wherein each construct comprises a gRNA sequence and a sensor sequence comprising the corresponding gRNA target sequence.
Expression-permitting culturing
Culturing the cells under conditions permitting the expression of the gRNA and the Cas9 protein.
PCR amplification of gRNA and sensor sequences
Amplifying the nucleic acid construct sequences by polymerase chain reaction (PCR) from the cells to obtain amplicons, wherein each amplicon comprises the gRNA sequence and the sensor sequence.
Sequencing amplicons containing gRNA and sensor
Sequencing the plurality of amplicons.
Determining sensor alteration patterns per amplicon
Determining a pattern of alterations in the sensor sequence in each of the amplicons.
Selecting gRNAs producing a desired sensor alteration pattern
Identifying a population of gRNAs that produce a desired pattern of alterations in the sensor sequence.
Electroporation delivery of Cas9 mRNA
Wherein the nucleic acid encoding the Cas9 protein mRNA is introduced into the population of cells by electroporation.
Across the independent claim, optimized gRNA selection is enabled by coupling each gRNA to a sensor sequence containing the corresponding gRNA target, expressing a Cas9 protein in the cell population, sequencing PCR amplicons that include both gRNA and sensor, determining sensor sequence alteration patterns, and selecting gRNAs that yield a desired pattern.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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