Tuning CRISPR/Cas9 activity with chemically modified nucleotide substitutions
AssigneesBoard of Trustees of Southern Illinois University, The Royal Institution for the Advancement of Learning/Mcgill University
InventorsKeith Gagnon, Masad Damha, Elise Malek-Adamian, Maryam Habibian, Daniel O'Reilly, Zachary Kartje
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Abstract
The present disclosure provides CRISPR/Cas9 ribonucleoprotein compositions comprising chemically modified CRISPR RNA (crRNA) guide and trans-acting CRISPR RNA (tracrRNA) components. Methods of using the disclosed CRISPR/Cas9 ribonucleoprotein compositions are also provided.
Claims24
A composition comprising a Cas9 protein, a first oligonucleotide comprising a first region having a 5′ end and a 3′ end and a second region, and a second oligonucleotide comprising a first region and a second region, wherein said second region of said first oligonucleotide interacts with said first region of said second oligonucleotide, and said second region of said second oligonucleotide interacts with said Cas9 protein,
- wherein said first oligonucleotide:
- a) comprises at least a first 2′-deoxyribonucleotide or 2′-deoxyribonucleotide analog in said second region or within about 12 nucleotides of said 5′ end of said first region;
- and
- b) comprises at least two ribonucleotides or ribonucleotide analogs within about 8 nucleotides of the 3′ end of said first region,
- wherein said second region of said first oligonucleotide comprises the chemical structure of crRNA-E2_J.
A composition comprising a Cas9 protein, a first oligonucleotide comprising a first region having a 5′ end and a 3′ end and a second region, and a second oligonucleotide comprising a first region and a second region, wherein said second region of said first oligonucleotide interacts with said first region of said second oligonucleotide, and said second region of said second oligonucleotide interacts with said Cas9 protein, wherein said first oligonucleotide:
- a) comprises at least a first 2′-deoxyribonucleotide or 2′-deoxyribonucleotide analog in said second region or within about 12 nucleotides of said 5′ end of said first region;
- and
- b) comprises at least two ribonucleotides or ribonucleotide analogs within about 8 nucleotides of the 3′ end of said first region,
- wherein said first oligonucleotide comprises the chemical structure of crRNA-E2_J.
Dependencies are inferred from the claim text.