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AssigneesThe Broad Institute, Inc., President and Fellows of Harvard College, Trustees of Boston University
InventorsDavid R. Liu, Tony P. Huang, Zachary J. Heins, Ahmad S. Khalil
Estimating the term…
The present disclosure provides Cas9 variants, and base editors comprising these variants, that recognize non-canonical protospacer adjacent motifs (PAMs) and have less restrictive PAM requirements for editing. The present disclosure provides Cas9 protein variants comprising one or more amino acid substitutions relative to wild-type Nme2Cas9. Fusion proteins comprising the Cas protein variants described herein are also provided by the present disclosure. Further provided herein are methods for editing a target nucleic acid using the Cas variants and fusion proteins provided herein. The present disclosure also provides guide RNAs, complexes, polynucleotides, cells, kits, and pharmaceutical compositions. Further described herein are phage-assisted continuous evolution (PACE) systems, vectors, methods, and devices.
A Cas protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a Cas protein of SEQ ID NO:
A guide RNA (gRNA) comprising, a nucleic acid sequence comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nucleic acid sequence of any one of the spacers in Table 2.
A complex comprising a fusion protein and the guide RNA of any one of claims A38-A44.
A vector system for phage-assisted continuous evolution comprising:
A method of continuous evolution comprising:
A vector comprising a nucleic acid coding sequence that encodes a gIII protein comprising an in cis split intein pair connected by a polynucleotide insert sequence, at least 1 protospacer sequence, and at least 1 PAM sequence.
One or more vectors comprising a nucleic acid sequence that encodes a fusion protein comprising a TadA8e domain and a dNme2Cas9 domain connected by a polynucleotide insert sequence and an in trans intein.
A method comprising transforming cells with a base editing (BE)-expressing plasmid (BP) and a library plasmid (LP), and further subjecting these cells to (a) an induction, (b) signal amplification, (c) harvesting, and (d) sequence analysis.
Dependencies are inferred from the claim text.