US 11,866,699 B2
Genome editing reagents and their use
Marc Joseph Lajoie, Seattle, WA (US); Elizabeth Erin Gray, Seattle, WA (US); Neil P. King, Seattle, WA (US); Daniel Brewster Stetson, Seattle, WA (US); David Baker, Seattle, WA (US); and Katherine DaPron, Seattle, WA (US)
Assigned to UNIVERSITY OF WASHINGTON, Seattle, WA (US)
Appl. No. 16/476,917
Filed by UNIVERSITY OF WASHINGTON, Seattle, WA (US)
PCT Filed Feb. 12, 2018, PCT No. PCT/US2018/017796
§ 371(c)(1), (2) Date Jul. 10, 2019,
PCT Pub. No. WO2018/148647, PCT Pub. Date Aug. 16, 2018.
Claims priority of provisional application 62/457,369, filed on Feb. 10, 2017.
Prior Publication US 2019/0352639 A1, Nov. 21, 2019
Int. Cl. C07H 21/04 (2006.01); C12N 15/11 (2006.01); C07K 14/005 (2006.01); C12N 9/22 (2006.01); C12N 15/85 (2006.01)
CPC C12N 15/11 (2013.01) [C07K 14/005 (2013.01); C12N 9/22 (2013.01); C12N 15/111 (2013.01); C12N 15/85 (2013.01); C12Y 301/21 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01); C12Q 2521/301 (2013.01)] 15 Claims
 
1. A composition comprising:
(a) a multimeric assembly, comprising a plurality of oligomeric substructures, wherein each oligomeric substructure comprises a plurality of recombinant polypeptides that self-interact around at least one axis of rotational symmetry, wherein
(i) each of the recombinant polypeptides comprises a polypeptide-polypeptide interface (“O interface”):
(ii) one or more of the recombinant polypeptides comprises a polypeptide domain that is capable of interacting with a lipid bilayer (“M domain”);
(iii) one or more of the recombinant polypeptides comprises a polypeptide domain that is capable of effecting membrane scission and release of an enveloped multimeric assembly from a cell by recruiting the ESCRT machinery to the site of budding by binding to one or more proteins in the eukaryotic ESCRT complex (“L domain”); and
(iv) one or more of the recombinant polypeptides comprises an RNA binding domain (“RBD”);
wherein the M domain, the L domain, the O interface, and the RBD are not each present in a single naturally occurring protein; and
(b) one or more active component selected from the group consisting of:
(i) one or more RNAs comprising
(A) one or more guide RNA (gRNA), wherein each gRNA comprises (I) a guide sequence capable of binding to a desired nucleic acid sequence; (II) an RNA packaging sequence capable of being bound by the RBD; and (III) a structural sequence capable of binding to a Class 2 Cas protein to form a Class 2 Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)-Cas ribonucleoprotein (RNP) complex; and
(B) one or more mRNA encoding a Class 2 Cas protein and an RNA packaging sequence capable of being bound by the RBD; and/or
(ii) one or more Class 2 CRISPR-Cas RNP complexes comprising a Class 2 Cas protein and one or more gRNA bound by the Class 2 Cas protein, wherein each gRNA comprises (I) a guide sequence capable of binding to a desired nucleic acid sequence; (II) an RNA packaging sequence capable of being bound by the RBD; and (III) a structural sequence capable of binding to a Class 2 Cas protein;
wherein the structural sequence comprises or consists of a sequence that is at least 50% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOS:332-335:
(SEQ ID NO: 332)
GUUUAAGAGCUA-N-UGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC,
 
(SEQ ID NO: 333)
GUUUUAGUACUCUG-N-GAAACAGAAUCUACUAAAACAAGGCAAAAUG
CCGUGUUUAUCUCGUCAACUUGUUGGCGAGA,
 
(SEQ ID NO: 334)
AAUUUCUACUC-N-UUGUAGAU,
and
 
(SEQ ID NO: 335)
AAUUUCUACUAA-N-GUGUAGAU;
wherein N is absent or comprises or consists of an RNA packaging sequence.