US 11,773,409 B2
CRISPR/Cas 9-mediated integration of polynucleotides by sequential homologous recombination of AAV donor vectors
Rasmus O. Bak, Risskov (DK); Matthew Porteus, Stanford, CA (US); and Sriram Vaidyanathan, Mountain View, CA (US)
Assigned to The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
Appl. No. 16/607,029
Filed by The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
PCT Filed Apr. 23, 2018, PCT No. PCT/US2018/028957
§ 371(c)(1), (2) Date Oct. 21, 2019,
PCT Pub. No. WO2018/195555, PCT Pub. Date Oct. 25, 2018.
Claims priority of provisional application 62/488,627, filed on Apr. 21, 2017.
Prior Publication US 2020/0131539 A1, Apr. 30, 2020
Int. Cl. C12N 15/00 (2006.01); C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 15/86 (2006.01); C12N 15/861 (2006.01); A61K 35/76 (2015.01); A61K 39/235 (2006.01); A61K 48/00 (2006.01); C07K 14/075 (2006.01)
CPC C12N 15/902 (2013.01) [A61K 35/76 (2013.01); C12N 9/22 (2013.01); C12N 15/86 (2013.01); C12N 15/8616 (2013.01); A61K 39/235 (2013.01); A61K 48/005 (2013.01); C07K 14/075 (2013.01); C12N 15/907 (2013.01); C12N 2310/20 (2017.05); C12N 2310/313 (2013.01); C12N 2710/10043 (2013.01); C12N 2750/14141 (2013.01); C12N 2800/40 (2013.01)] 19 Claims
OG exemplary drawing
 
1. A system for CRISPR/Cas9-mediated integration of a target polynucleotide into a target genetic locus in a cell comprising:
(a) a first targeting AAV vector comprising a single guide RNA (sgRNA) target site with a protospacer-adjacent motif (PAM), a first donor template, a 5′ homology arm that is homologous to a first portion of the target locus, and a 3′ homology arm that is homologous to a second portion of the target locus that is not overlapping with the first portion of the target locus,
wherein the sgRNA target site is recognized by a target locus-specific sgRNA, wherein the first donor template comprises a first nucleotide sequence of the target polynucleotide;
(b) a second targeting AAV vector comprising a second donor template, a 5′ homology arm that is homologous to a first portion of the first donor template, a 3′ homology arm that is homologous to a second portion of the first targeting AAV vector,
wherein the first portion of the first donor template and the second portion of the first targeting AAV vector are not overlapping, the second donor template comprises a second nucleotide sequence of the target polynucleotide, and the nucleotide sequence of the target polynucleotide is split between the first donor template and the second donor template;
(c) the target locus-specific sgRNA; and
(d) a CRISPR-associated protein 9 (Cas9) polypeptide or a polynucleotide encoding the Cas9 polypeptide.