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Publication Number
US-11957114-B2
Publication Date
2024-04-16
Expiration Date
Abstract
Provided herein are systems and methods for Inducible and conditional CRISPR/Cas9 and RNAi. From animal model creation and the efficiency of CRISPR-based targeting, the present invention comprises developing RNAi models that enable inducible and reversible gene silencing to simulate new therapeutic regimes.
Core Innovation
The invention provides a method of using founder knock-in strains to enable generation of subsequent RNAi strains in rat embryos or cells. A founder strain is created by inserting, using a CRISPR/Cas9 system, a nucleotide sequence comprising a promoter sequence operably linked to a miRNA backbone sequence and a reporter sequence downstream of the Col1a1 gene on chromosome 10 of rat.
After creation of the founder strain, embryos or cells produced using the founder strain are obtained and transduced with a CRISPR/Cas9 system comprising a Cas9 protein, a gRNA targeted to the miRNA backbone sequence, and a donor template including a shRNA sequence. This transducing step integrates the shRNA sequence into the miRNA backbone sequence of the embryo or cell, producing a subsequent founder knock-in strain rat or cell in which the miRNA backbone sequence comprises the integrated shRNA sequence.
The platform is described as an inducible, conditional CRISPR/Cas9 plus RNAi system for creating rat RNAi models with reversible gene silencing. It includes using founders as landing pads to later knock in variable shRNA sequences into the miRNA backbone via CRISPR HDR, and discusses inducible and conditional systems including Tet/TRE and transactivator concepts.
Claims Coverage
The independent claim covers a two-stage use of founder knock-in strains to integrate promoter/miRNA backbone/reporter sequences at the Col1a1 locus and then use the founder as a landing pad to integrate variable shRNA sequences into the miRNA backbone via CRISPR/Cas9-mediated transduction. Dependent claims further refine the inventive concept with common sequences across subsequent strains, endogenous gene targeting by the variable shRNA, selectable genome editing system options, CRISPR/Cas9 component specification, promoter/enhancer options, and a rat source constraint for embryos or cells.
Founder knock-in at Col1a1 with promoter-miRNA backbone-reporter
Creating a founder strain rat whose genome comprises a promoter sequence operably linked to a miRNA backbone sequence and a reporter sequence downstream of the Col1a1 gene on chromosome 10, by using a CRISPR/Cas9 system to insert the nucleotide sequence comprising said promoter sequence operably linked to said miRNA backbone sequence and said reporter sequence downstream of the Col1a1 gene on chromosome 10.
CRISPR/Cas9 transducing to integrate shRNA into the miRNA backbone
Obtaining an embryo or a cell produced using the founder strain and transducing the embryo or cell with a CRISPR/Cas9 system comprising a Cas9 protein, a gRNA targeted to the miRNA backbone sequence, and a donor template that includes a shRNA sequence, thereby inserting said shRNA sequence into said miRNA backbone sequence to produce a subsequent founder knock-in strain rat or cell wherein the miRNA backbone sequence comprises the integrated shRNA sequence.
Using the founder strain to knock in variable shRNA for subsequent strains
Using the founder strain to knock in a variable shRNA sequence for a subsequent strain to be produced.
Common sequences across subsequent RNAi strains
The method creates a founder strain using a genome editing system to insert common sequences for each subsequent RNAi strain, and uses the founder strain to generate subsequent strains by transduction with shRNA targeting an endogenous gene using a variable shRNA sequence.
Endogenous gene targeted by variable shRNA
The variable shRNA targets an endogenous gene in the subsequent RNAi strain generation.
Selectable gene editing system options
The gene editing system is selected from the group consisting of a CRISPR/Cas9 system, zinc finger nucleases, and transcription activator-like effector nucleases.
CRISPR/Cas9 system components include Cas9, gRNA, and donor template
The CRISPR/Cas9 system includes a Cas9 protein, a gRNA, and a donor template.
Promoter selection for miRNA-based shRNA expression
The promoter driving expression of the miRNA-based shRNA is selected from a specified group of inducible and constitutive promoter/enhancer systems compatible with the host cell systems.
Rat source for embryos or cells
The embryos or cells are from the rat species.
Overall, the claim coverage centers on a two-stage founder knock-in strain platform: first integrating a promoter/miRNA backbone/reporter construct into the Col1a1 locus via CRISPR/Cas9, and then using embryos or cells derived from that founder as landing pads for CRISPR/Cas9 transduction to integrate variable shRNA sequences into the miRNA backbone. Dependent claims further specify shared/common sequence structure across strains, endogenous gene targeting by the variable shRNA, selectable nucleases and CRISPR components, promoter/enhancer options, and that the embryos or cells are rat-derived.
Stated Advantages
Documented Applications
No documented applications found
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