Synthetic miniature Crispr-Cas (CasMINI) system for eukaryotic genome engineering
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Publication Number
US-12415994-B2
Publication Date
2025-09-16
Expiration Date
Abstract
Provided herein are Cas proteins and guide RNA molecules engineered to exhibit increased activity in eukaryotic cells. The provided Cas proteins and RNA molecules are particularly useful for applications where modulation of eukaryotic nucleic acids with relatively small molecules is advantageous. Also provided are nucleic acids and vectors encoding the disclosed Cas proteins and guide RNA molecules, pharmaceutical compositions including the Cas proteins and guide RNA molecules, and methods for using the disclosed materials.
Core Innovation
The disclosed invention provides a synthetic miniature CRISPR-Cas (CasMINI) system for eukaryotic genome engineering, including engineered compact Cas proteins for modulating a target nucleic acid in eukaryotic cells. The CasMINI proteins are engineered from type V-F Cas14 (Cas14) and are described as miniature Cas proteins about 529 amino acids derived from the native Cas14 sequence (SEQ ID NO: 1).
The invention also provides engineered sgRNAs, including crRNA/tracrRNA fusion designs, with modifications to poly-U/poly-G regions and 3′ stem-loop truncations and/or 5′ poly-G deletions. The described Cas engineering includes a conserved (D/E/K/N)X(R/F)(E/K)N motif (SEQ ID NO: 253) and an at least 80% sequence identity requirement to wild-type Cas, together with substitution-position rules using specified positions including 241 and/or 516 and electrically charged residue positions.
The system further provides nucleic acids, vectors, and systems comprising the engineered Cas and sgRNA, and pharmaceutical compositions and therapeutic uses. The document reports iterative protein engineering and sgRNA optimization yielding large improvements in gene activation, no detectable off-targets, functional endogenous gene activation with dCasMINI-VPR, robust A·T-to-G·C base editing when fused to an adenine base editor (ABE8e), and transcriptional repression using dCasMINI with KRAB plus indel-mediated editing with selected nuclease-active CasMINI variants.
Claims Coverage
The provided claim set includes two independent claims. Both independent claims cover an engineered CRISPR-associated Cas protein capable of modulating a target nucleic acid in eukaryotic cells, optionally with an effector domain attached, and both require sequence identity to a native Cas sequence with specified substitution constraints; one independent claim additionally limits substitution counts around specified sequence regions and positions.
Engineered Cas protein with native-like sequence identity and specified substitution positions
An engineered CRISPR-associated Cas protein with an amino acid sequence of a native amino acid sequence of SEQ ID NO: 1, at least 95% identical to SEQ ID NO: 1, with one or more substitutions, where the substitutions comprise at least one substitution at a position selected from D143, K11, K73, T147, E151, K154, E241, D318, K330, K457, E425, E462, N504, E507, N516, N519, E527, and E528, optionally with an effector domain attached.
Engineered Cas protein with conserved motif and constrained substitution regions near motif and key positions
An engineered CRISPR-associated Cas protein with an amino acid sequence at least 95% identical to SEQ ID NO: 1, wherein the native sequence comprises a (D/E/K/N)X(R/F)(E/K)N motif (SEQ ID NO: 253), and where the substitutions comprise at least one of no more than six substitutions within or no more than 30 amino acids upstream or downstream of the motif, no more than six substitutions within or no more than 30 amino acids upstream or downstream of position 241, and/or no more than six substitutions within or no more than 30 amino acids upstream or downstream of position 516, and where the substitutions comprise at least one substitution at a position selected from D143, T147, E151, K154, E241, N504, E507, N516, N519, E527, and E528, optionally with an effector domain attached.
Across the independent claims, the core scope is an engineered Cas protein that remains highly identical to SEQ ID NO: 1 while introducing defined substitutions at specified residue positions and, in one independent claim, restricting substitution density around the conserved SEQ ID NO: 253 motif and around positions 241 and/or 516; both claims allow an effector domain to be attached.
Stated Advantages
Improved gene activation by 2–3 logs with no detectable off-targets.
Functional endogenous gene activation using dCasMINI-VPR.
Robust A·T-to-G·C base editing when fused to ABE8e.
Transcriptional repression using dCasMINI-KRAB and indel-mediated editing using selected nuclease-active CasMINI variants.
Comparable or better performance versus larger Cas12a for activation, as reported.
Whole-transcriptome RNA-seq showing similar specificity.
Documented Applications
Eukaryotic genome engineering using the CasMINI system to modulate target nucleic acids, including gene activation of endogenous genes.
Therapeutic use, supported by the presence of pharmaceutical compositions and therapeutic uses in the document.
Gene activation and repression applications including dCasMINI-VPR and dCasMINI-KRAB.
Base editing application using ABE8e fusion for A·T-to-G·C base editing.
Indel-mediated editing application using selected nuclease-active CasMINI variants.
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