Degenerate CRISPR Cas13a crRNAs for Detection of Highly Variable RNA Sequences
AssigneeThe Government of the United States of America, as represented by the Secretary of the Navy
InventorsTomasz A. Leski, Joseph R. Spangler, Zheng Wang, Scott N. Dean, David A. Stenger
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Abstract
A technique for the design of minimum CRISPR RNA (crRNA) sets aids in the detection of diverse nucleic acid targets using sequence degeneracy. As a working example, candidate degenerate Cas13a crRNA sets were designed for detection of diverse RNA targets (Lassa virus). A decision tree machine learning (ML) algorithm (RuleFit) was applied to define the top attributes that determine the specificity of degenerate crRNAs to elicit collateral nuclease activity. This general ML approach can be applied to the design of degenerate crRNA sets for any CRISPR/Cas system.
Claims12
A method for detecting members of a phylogenetically diverse group, the method comprising:
- (1) identifying conserved regions in a set of diverse nucleic acids in the phylogenetically diverse group;
- (2) designing candidate degenerate complementary spacer regions of CRISPR guide RNAs (crRNAs) corresponding to the conserved regions;
- (3) conducting high-throughput screening of the candidate degenerate crRNAs against complementary synthetic targets to obtain high performing degenerate crRNAs;
- (4) conducting high-throughput screening of the high performing degenerate crRNAs against targets representing at least a majority of the phylogenetically diverse group and at least one target representing a near neighbor to the phylogenetically diverse group to obtain a dataset;
- and
- (5) using a machine learning algorithm to analyze the dataset to identify generalizable crRNA design rules for detection of members of the phylogenetically diverse group.
A CRISPR guide RNA comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:
- 3 through 141, inclusive.
A Cas-based assay system comprising CRISPR guide RNA comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:
- 3 through 141, inclusive.
Dependencies are inferred from the claim text.