Granted Patent
B2
US 12,612,633 · App. 19/002,832 · Granted Apr 28, 2026
Compositions for inducing modifications of target endogenous nucleic acid sequences in nucleuses of eukaryotic cells
Assignee:
ToolGen Incorporated
C12N15/52C12N9/16C12N9/22C12N15/102C12N15/111C12N15/63C12N15/8216C12N15/85C12N15/907C12Y301/21C12N2310/10C12N2310/20C12N2310/531
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Abstract
The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides for compositions and methods that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. For example, disclosed herein are methods of modifying a target endogenous nucleic acid sequence in a nucleus of a eukaryotic cell in vitro or ex vivo, comprising: preparing a composition comprising a recombinant Cas9 protein, a single guide RNA, and a Cas9/RNA complex formed by at least a part of said recombinant Cas9 protein and single guide RNA, and introducing a Cas9/RNA complex into the eukaryotic cell in vitro or ex vivo, wherein in the composition, the single guide RNA is at least two-fold molar excess over the recombinant Cas9 protein, wherein the Cas9/RNA complex is a combination of the recombinant Cas9 protein and the single guide RNA, and wherein the Cas9/RNA complex is complexed prior to being introduced into the eukaryotic cell; wherein the single guide RNA is in vitro transcribed RNA or synthetic RNA, wherein the combination of the recombinant Cas9 protein and the single guide RNA produces a modification of the target endogenous nucleic acid sequence in the nucleus of the eukaryotic cell.
Claims (28)
1 . A method of modifying a target endogenous nucleic acid sequence in a nucleus of a eukaryotic cell, comprising:
providing a composition comprising an amount of a recombinant Cas9 protein and an amount of a single-guide RNA (sgRNA) to an in vitro environment,
wherein the recombinant Cas9 protein comprises a nuclear localization signal (NLS);
wherein the amount of the sgRNA is present in the composition in at least a two-fold molar excess over the amount of the recombinant Cas9 protein, and
wherein a Cas9/sgRNA complex forms in the in vitro environment prior to the composition being introduced into the eukaryotic cell; and
introducing the composition comprising the Cas9/sgRNA complex into the eukaryotic cell in vitro or ex vivo, wherein the Cas9/sgRNA complex produces a modification of the target endogenous nucleic acid sequence in the nucleus of the eukaryotic cell.
2 . The method of claim 1 , wherein the composition comprising the Cas9/sgRNA complex is introduced into the eukaryotic cell by transfection, wherein the transfection is performed by the method selected from the group consisting of electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain mediated transduction, and polyethylene glycol (PEG)-mediated transfection of protoplasts.
3 . The method of claim 2 , wherein the composition comprising the Cas9/sgRNA complex is introduced into the eukaryotic cell by electroporation.
4 . The method of claim 1 , wherein the target endogenous nucleic acid comprises a trinucleotide protospacer adjacent motif (PAM) recognized by the recombinant Cas9 protein, wherein the PAM consists of trinucleotide 5′-NGG-3′.
5 . The method of claim 1 , wherein the NLS is at the C-terminus of the recombinant Cas9 protein.
6 . The method of claim 1 , wherein the modification includes any one of a deletion, insertion, or substitution of at least one nucleotide.
7 . The method of claim 1 , further comprising inducing divisions of the eukaryotic cell to be divided into a plurality of cells which include the modified nucleic acid sequence.
8 . The method of claim 1 , wherein the sgRNA and recombinant Cas9 protein are present in a molar ratio from 29:1.4 to 29:14.
9 . The method of claim 1 , wherein the eukaryotic cell is a mammalian cell.
10 . The method of claim 1 , wherein the eukaryotic cell is a human cell.
11 . The method of claim 1 , wherein the recombinant Cas9 protein is derived from a Streptococcus pyogenes Cas9 protein.
12 . The method of claim 1 , wherein the recombinant Cas9 protein is a recombinant Cas9 protein expressed in E. coli.
13 . The method claim 1 , wherein the single-guide RNA comprises a CRISPR RNA (crRNA) fused to a trans-activating crRNA (tracrRNA), and wherein the crRNA comprises a sequence complementary the target endogenous nucleic acid sequence.
14 . The method of claim 1 , wherein the sgRNA and recombinant Cas9 protein are present in a molar ratio from 2:1 to 29:4.5.
15 . The method of claim 1 , wherein the sgRNA and recombinant Cas9 protein are present in a weight ratio of 8:15 to 20:15.
16 . A method of modifying a target endogenous nucleic acid sequence in a nucleus of a human cell, comprising:
providing a composition comprising an amount of a recombinant Cas9 protein and an amount of a single-guide RNA (sgRNA) to an in vitro environment,
wherein the recombinant Cas9 protein comprises a nuclear localization signal (NLS);
wherein the amount of the sgRNA is present in the composition in at least a two-fold molar excess over the amount of the recombinant Cas9 protein, and
wherein a Cas9/sgRNA complex forms in the in vitro environment prior to the composition being introduced into the human cell; and
introducing the composition comprising the Cas9/sgRNA complex into the human cell in vitro or ex vivo via electroporation, wherein the Cas9/sgRNA complex produces a modification of the target endogenous nucleic acid sequence in the nucleus of the human cell.
17 . The method of claim 16 , wherein the sgRNA and the recombinant Cas9 protein are present in a molar ratio from 2:1 to 29:4.5.
18 . The method of claim 16 , wherein the sgRNA and the recombinant Cas9 protein are present in a weight ratio of 8:15 to 20:15.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Oct 22, 2025
From: KIM, JIN-SOO; CHO, SEUNG WOO; KIM, SOJUNG
To: TOOLGEN INCORPORATED
Reel/Frame 072632/0861 →
Continuity (8)
Continuation
PCTKR2013009488
· Oct 23, 2013
Provisional Application
61837481
· Jun 20, 2013
Provisional Application
61803599
· Mar 20, 2013
Provisional Application
61717324
· Oct 23, 2012
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