IP Library › Granted Patent US 12,553,064
Granted Patent B2
US 12,553,064 · App. 17/713,696 · Granted Feb 17, 2026

Methods and compositions for targeted genetic modification through single-step multiple targeting

Inventors: Vera Voronina (North Bethesda, MD); Lynn Macdonald (Harrison, NY); Brian Zambrowicz (Sleepy Hollow, NY); Andrew J. Murphy (Croton-on-Hudson, NY)
Assignee: Regeneron Pharmaceuticals, Inc.
C12N15/907A01K67/0276A01K67/0278A01K2217/072A01K2217/075A01K2227/105C12N9/22C12N15/8509
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,553,064
App. No.
17/713,696
Granted
Feb 17, 2026
Kind
B2
Abstract

Methods and compositions are provided for making one or more targeted genetic modifications at a target genomic locus within a cell and for producing non-human animals comprising the modified genomic locus. The methods employ two or more large targeting vectors which are capable of recombining with each other and with the target genomic locus in a single genomic targeting step. The methods may also be employed in combination with a nuclease agent. Methods and compositions are also provided for enhancing homologous recombination at a target genomic locus in a cell. The methods employ two or more nucleic acids comprising one or more overlapping sequences. The methods may be employed in combination with a nuclease agent or without a nuclease agent.

Claims (55)

1 . A method for modifying a target genomic locus on an endogenous chromosome in a mouse embryonic stem (ES) cell, comprising:

(I) (a) introducing into the mouse ES cell a nuclease agent or one or more polynucleotides encoding the nuclease agent, wherein the nuclease agent is a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN), or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein and a guide RNA (gRNA), and wherein the nuclease agent makes a single or double-strand break within the target genomic locus;

(b) introducing into the mouse ES cell a first large targeting vector (LTVEC) that comprises a first nucleic acid insert flanked by a first 5′ homology arm and a first 3′ homology arm, and a second LTVEC that comprises a second nucleic acid insert flanked by a second 5′ homology arm and a second 3′ homology arm,

wherein the sum total of the first 5′ homology arm and the first 3′ homology arm of the first LTVEC is from 10 kb to 150 kb, and the sum total of the second 5′ homology arm and the second 3′ homology arm of the second LTVEC is from 10 kb to 150 kb,

wherein the first 5′ homology arm of the first LTVEC is at least 5 kb, and the second 3′ homology arm of the second LTVEC is at least 5 kb,

wherein the combined size of the first nucleic acid insert and the second nucleic acid insert is from 100 kb to 500 kb,

wherein the first nucleic acid insert and/or the second nucleic acid insert comprises a nucleic acid encoding a selection marker,

wherein the first 3′ homology arm of the first LTVEC has a first overlapping sequence with the second 5′ homology arm of the second LTVEC, wherein the size of the first overlapping sequence is from 10 kb to 70 kb, and wherein the first 3′ homology arm of the first LTVEC is identical to the second 5′ homology arm of the second LTVEC, and

wherein the first 5′ homology arm of the first LTVEC is homologous to a corresponding 5′ target sequence within the target genomic locus, and the second 3′ homology arm of the second LTVEC is homologous to a corresponding 3′ target sequence within the target genomic locus,

wherein the method does not comprise introducing a separate expression construct comprising a selection marker not in conjunction with the first LTVEC or the second LTVEC, and

wherein the target genomic locus is modified by integration of the first nucleic acid insert and the second nucleic acid insert by homologous recombination between the first 5′ homology arm of the first LTVEC and the 5′ target sequence within the target genomic locus, between the first 3′ homology arm of the first LTVEC and the second 5′ homology arm of the second LTVEC, and between the second 3′ homology arm of the second LTVEC and the 3′ target sequence within the target genomic locus; and

(c) using the selection marker encoded by the nucleic acid in the first nucleic acid insert and/or the second nucleic acid insert to select a targeted mouse ES cell comprising the first nucleic acid insert and the second nucleic acid insert integrated into the target genomic locus; or

(II) (a) introducing into the mouse ES cell a nuclease agent or one or more polynucleotides encoding the nuclease agent, wherein the nuclease agent is a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN), or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein and a guide RNA (gRNA), and wherein the nuclease agent makes a single or double-strand break within the target genomic locus;

(b) introducing into the mouse ES cell a first large targeting vector (LTVEC) that comprises a first nucleic acid insert flanked by a first 5′ homology arm and a first 3′ homology arm, a second LTVEC that comprises a second nucleic acid insert flanked by a second 5′ homology arm and a second 3′ homology arm, and a third LTVEC that comprises a third nucleic acid insert flanked by a third 5′ homology arm and a third 3′ homology arm,

wherein the sum total of the first 5′ homology arm and the first 3′ homology arm of the first LTVEC is from 10 kb to 150 kb, the sum total of the second 5′ homology arm and the second 3′ homology arm of the second LTVEC is from 10 kb to 150 kb, and the sum total of the third 5′ homology arm and the third 3′ homology arm of the third LTVEC is from 10 kb to 150 kb,

wherein the first 5′ homology arm of the first LTVEC is at least 5 kb, and the third 3′ homology arm of the third LTVEC is at least 5 kb,

wherein the combined size of the first nucleic acid insert, the second nucleic acid insert, and the third nucleic acid insert from 100 kb to 700 kb,

wherein the first nucleic acid insert, the second nucleic acid insert, and/or the third nucleic acid insert comprises a nucleic acid encoding a selection marker,

wherein the first 3′ homology arm of the first LTVEC has a first overlapping sequence with the second 5′ homology arm of the second LTVEC, the second 3′ homology arm of the second LTVEC has a second overlapping sequence with the third 5′ homology arm of the third LTVEC, and the first 5′ homology arm of the first LTVEC is homologous to a corresponding 5′ target sequence within the target genomic locus, and the third 3′ homology arm of the third LTVEC is homologous to a corresponding 3′ target sequence within the target genomic locus,

wherein the first 3′ homology arm of the first LTVEC is identical to the second 5′ homology arm of the second LTVEC, and wherein the second 3′ homology arm of the second LTVEC is identical to the third 5′ homology arm of the third LTVEC,

wherein the size of the first overlapping sequence is from 10 kb to 70 kb, and wherein the size of the second overlapping sequence is from 10 kb to 70 kb,

wherein the method does not comprise introducing a separate expression construct comprising a selection marker not in conjunction with the first LTVEC, the second LTVEC, or the third LTVEC, and

wherein the target genomic locus is modified by integration of the first nucleic acid insert, the second nucleic acid insert, and the third nucleic acid insert by homologous recombination between the first 5′ homology arm of the first LTVEC and the 5′ target sequence within the target genomic locus, between the first 3′ homology arm of the first LTVEC and the second 5′ homology arm of the second LTVEC, between the second 3′ homology arm of the second LTVEC and the third 5′ homology arm of the third LTVEC, and between the third 3′ homology arm of the third LTVEC and the 3′ target sequence within the target genomic locus; and

(c) using the selection marker encoded by the nucleic acid in the first nucleic acid insert, the second nucleic acid insert, and/or the third nucleic acid insert to select a targeted mouse ES cell comprising the first nucleic acid insert, the second nucleic acid insert, and the third nucleic acid insert integrated into the target genomic locus.

2 . The method of claim 1 , wherein the first LTVEC and the second LTVEC in option (I) comprise overlapping fragments of a contiguous nucleic acid, which is reformed by integration of the first nucleic acid insert and the second nucleic acid insert into the target genomic locus; or

wherein the first LTVEC, the second LTVEC, and the third LTVEC in option (II) comprise overlapping fragments of a contiguous nucleic acid, which is reformed by integration of the first nucleic acid insert, the second nucleic acid insert, and the third nucleic acid insert into the target genomic locus.

3 . The method of claim 1 , wherein the nuclease agent is the ZFN or the TALEN.

4 . The method of claim 1 , wherein the nuclease agent comprises the Cas protein and the gRNA.

5 . The method of claim 4 , wherein the Cas protein is Cas9.

6 . The method of claim 1 , wherein the first nucleic acid insert, the second nucleic acid insert, or both in option (I) are from a species that is different from the species of the mouse ES cell; or

wherein one or more of the first nucleic acid insert, the second nucleic acid insert, and the third nucleic acid insert in option (II) are from a species that is different from the species of the mouse ES cell.

7 . The method of claim 6 , wherein the first nucleic acid insert, the second nucleic acid insert, or both in option (I) are human nucleic acids; or

wherein one or more of the first nucleic acid insert, the second nucleic acid insert, and the third nucleic acid insert in option (II) are human nucleic acids.

8 . The method of claim 1 , wherein integration of the first nucleic acid insert, the second nucleic acid insert, or both into the target genomic locus in option (I) or wherein integration of one or more of the first nucleic acid insert, the second nucleic acid insert, and the third nucleic acid insert into the target genomic locus in option (II) results in one or more of:

(a) an addition of an exogenous sequence at the target genomic locus;

(b) a deletion of an endogenous sequence at the target genomic locus; and

(c) a knock-in, a knockout, a point mutation, a domain swap, an exon swap, an intron swap, a regulatory sequence swap, a gene swap, or a combination thereof.

9 . The method of claim 8 , wherein integration of the first nucleic acid insert, the second nucleic acid insert, or both into the target genomic locus in option (I) results in the deletion of the endogenous sequence at the target genomic locus, wherein the deletion is from about 5 kb to about 800 kb; or

wherein integration of one or more of the first nucleic acid insert, the second nucleic acid insert, and the third nucleic acid insert into the target genomic locus in option (II) results in the deletion of the endogenous sequence at the target genomic locus, wherein the deletion is from about 5 kb to about 800 kb.

10 . The method of claim 1 , wherein the size of the first overlapping sequence in option (I) is at least 20 kb; or

wherein the size of the first overlapping sequence is at least 20 kb, and/or the size of the second overlapping sequence in option (II) is at least 20 kb.

11 . The method of claim 1 , wherein the combined size of the first nucleic acid insert and the second nucleic acid insert in option (I) is at least 200 kb; or

wherein the combined size of the first nucleic acid insert, the second nucleic acid insert, and the third nucleic acid insert in option (II) is at least 200 kb.

12 . The method of claim 1 , wherein the combined size of the first nucleic acid insert and the second nucleic acid insert in option (I) is at least 300 kb; or

wherein the combined size of the first nucleic acid insert, the second nucleic acid insert, and the third nucleic acid insert in option (II) is at least 400 kb.

13 . The method of claim 1 , wherein the first LTVEC is at least 50 kb and the second LTVEC is at least 50 kb in option (I); or

wherein the first LTVEC is at least 50 kb, the second LTVEC is at least 50 kb, and the third LTVEC is at least 50 kb in option (II).

14 . The method of claim 1 , wherein the first LTVEC is at least 100 kb and the second LTVEC is at least 100 kb in option (I); or

wherein the first LTVEC is at least 100 kb, the second LTVEC is at least 100 kb, and the third LTVEC is at least 100 kb in option (II).

15 . The method of claim 1 , wherein the first LTVEC is a linear nucleic acid and/or the second LTVEC is a linear nucleic acid in option (I); or

wherein the first LTVEC is a linear nucleic acid, and/or the second LTVEC is a linear nucleic acid, and/or the third LTVEC is a linear nucleic acid in option (II).

16 . The method of claim 1 , wherein the targeting efficiency is at least 0.4%.

17 . A method for producing an F0 generation mouse, comprising:

(a) introducing a mouse ES cell into a mouse host embryo, wherein the mouse ES cell was produced by the method of claim 1 ; and

(b) gestating the mouse host embryo in a mouse surrogate mother, wherein the mouse surrogate mother produces the F0 generation mouse.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 21, 2025
From: VORONINA, VERA; MACDONALD, LYNN; ZAMBROWICZ, BRIAN; MURPHY, ANDREW J.
To: REGENERON PHARMACEUTICALS, INC.
Reel/Frame 070583/0870 →
Continuity (5)
Continuation 14974623 · Dec 18, 2015
Provisional Application 62205524 · Aug 14, 2015
Provisional Application 62167408 · May 28, 2015
Provisional Application 62094104 · Dec 19, 2014
Related Publication 20220235381A1 · Jul 28, 2022
References Cited (226)
US 5612205A · Kay et al. · 1997 [cited by applicant]
US 6372956B1 · Goldsmith et al. · 2002 [cited by applicant]
US 6566579B1 · Jaisser et al. · 2003 [cited by applicant]
US 6586251B2 · Economides et al. · 2003 [cited by applicant]
US 7105348B2 · Murphy et al. · 2006 [cited by applicant]
US 7294754B2 · Poueymirou et al. · 2007 [cited by applicant]
US 7771967B2 · Huang et al. · 2010 [cited by applicant]
US 8502018B2 · Murphy et al. · 2013 [cited by applicant]
US 8697359B1 · Zhang · 2014 [cited by applicant]
US 9228208B2 · Frendewey et al. · 2016 [cited by applicant]
US 9476065B2 · Horwitz et al. · 2016 [cited by applicant]
US 10362771B2 · Mashimo et al. · 2019 [cited by applicant]
US 11326184B2 · Voronina et al. · 2022 [cited by applicant]
US 20030134318A1 · Case et al. · 2003 [cited by applicant]
US 20030175968A1 · Golic et al. · 2003 [cited by applicant]
US 20040018626A1 · Murphy et al. · 2004 [cited by applicant]
US 20040197317A1 · Rao et al. · 2004 [cited by applicant]
US 20050144655A1 · Economides et al. · 2005 [cited by applicant]
US 20080066197A1 · Ying et al. · 2008 [cited by applicant]
US 20080113437A1 · Joly et al. · 2008 [cited by applicant]
US 20080299580A1 · DeKelver et al. · 2008 [cited by applicant]
US 20090055943A1 · Economides et al. · 2009 [cited by applicant]
US 20100218264A1 · Cui et al. · 2010 [cited by applicant]
US 20110207221A1 · Cost et al. · 2011 [cited by applicant]
US 20110307968A1 · Auerbach et al. · 2011 [cited by applicant]
US 20120272349A1 · Ochiya et al. · 2012 [cited by applicant]
US 20120315670A1 · Jacobson et al. · 2012 [cited by applicant]
US 20130210681A1 · Zhang et al. · 2013 [cited by applicant]
US 20130309670A1 · Frendewey et al. · 2013 [cited by applicant]
US 20140068797A1 · Doudna et al. · 2014 [cited by applicant]
US 20140273226A1 · Wu · 2014 [cited by applicant]
US 20140273234A1 · Zhang et al. · 2014 [cited by applicant]
US 20140301990A1 · Gregory et al. · 2014 [cited by applicant]
US 20140309487A1 · Lee et al. · 2014 [cited by applicant]
US 20140310828A1 · Lee et al. · 2014 [cited by applicant]
US 20140342456A1 · Mali et al. · 2014 [cited by applicant]
US 20150079680A1 · Bradley et al. · 2015 [cited by applicant]
US 20150140664A1 · Byrne et al. · 2015 [cited by applicant]
US 20150159174A1 · Frendewey et al. · 2015 [cited by applicant]
US 20150159175A1 · Frendewey et al. · 2015 [cited by applicant]
US 20150184199A1 · Horwitz et al. · 2015 [cited by applicant]
US 20150376650A1 · Auerbach et al. · 2015 [cited by applicant]
US 20150376651A1 · Frendewey et al. · 2015 [cited by applicant]
US 20160024529A1 · Carstens · 2016 [cited by applicant]
US 20160046960A1 · Frendewey et al. · 2016 [cited by applicant]
US 20160060657A1 · Frendewey et al. · 2016 [cited by applicant]
US 20160081314A1 · Thurston et al. · 2016 [cited by applicant]
US 20160108369A1 · Kuno et al. · 2016 [cited by applicant]
US 20160145646A1 · Frendewey et al. · 2016 [cited by applicant]
US 20160177339A1 · Voronina et al. · 2016 [cited by applicant]
US 20170251647A1 · Mashimo et al. · 2017 [cited by applicant]
US 20190338274A1 · Bradley et al. · 2019 [cited by applicant]
CN 1484707A · 2004 [cited by applicant]
EP 1360287B1 · 2012 [cited by applicant]
EP 3009511A2 · 2016 [cited by applicant]
EP 2596101B1 · 2018 [cited by applicant]
WO WO2002066630A1 · 2002 [cited by applicant]
WO WO2003087341A2 · 2003 [cited by applicant]
WO WO2006044962A1 · 2006 [cited by applicant]
WO WO2007117410A2 · 2007 [cited by applicant]
WO WO2008151081A1 · 2008 [cited by applicant]
WO WO2009104094A2 · 2009 [cited by applicant]
WO WO2011051390A1 · 2011 [cited by applicant]
WO WO2011078665A1 · 2011 [cited by applicant]
WO WO2011154927A2 · 2011 [cited by applicant]
WO WO2012012667A2 · 2012 [cited by applicant]
WO WO2012018726A1 · 2012 [cited by applicant]
WO WO2012129198A1 · 2012 [cited by applicant]
WO WO2013063361A1 · 2013 [cited by applicant]
WO WO2013163394A1 · 2013 [cited by applicant]
WO WO2013169802A1 · 2013 [cited by applicant]
WO WO2013176772A1 · 2013 [cited by applicant]
WO WO2014093908A2 · 2014 [cited by applicant]
WO WO2014104878A1 · 2014 [cited by applicant]
WO WO2014143381A1 · 2014 [cited by applicant]
WO WO2014172489A2 · 2014 [cited by applicant]
WO WO2015040402A1 · 2015 [cited by applicant]
WO WO2015077290A2 · 2015 [cited by applicant]
WO WO2015088643A1 · 2015 [cited by applicant]
WO WO2015095804A1 · 2015 [cited by applicant]
WO WO2015163733A1 · 2015 [cited by applicant]
WO WO2015166272A2 · 2015 [cited by applicant]
WO WO2015188109A1 · 2015 [cited by applicant]
WO WO2015200805A2 · 2015 [cited by applicant]
WO WO2016061073A1 · 2016 [cited by applicant]
WO WO2016061374A1 · 2016 [cited by applicant]
WO WO2016081923A2 · 2016 [cited by applicant]
WO WO2016089866A1 · 2016 [cited by applicant]
WO WO2016100819A1 · 2016 [cited by applicant]
Zhang et al., “The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation,” Plant Biotechnol. J., vol. 12(6), pp. 797-807, May 23, 2014. [cited by applicant]
“Stem Cells: Scientific Progress and Future Research Directions,” National Institute of Health, Department of Health and Human Services, (2001). [cited by applicant]
Abudayyeh et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science, vol. 353(6299), Jun. 2, 2016. [cited by applicant]
Auerbach et al., “Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines,” Biotechniques, vol. 29(5), pp. 1024-1028, 1030, 1032, Nov. 2000. [cited by applicant]
Benders et al., “Cloning whole bacterial genomes in yeast,” Nucleic Acids Res., vol. 38(8), pp. 2558-2569, Mar. 7, 2010. [cited by applicant]
Berdien, et al., “TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer,” Gene Therapy, 21, 539-548 (2014). [cited by applicant]
Bernardini et al., “Site-specific genetic engineering of the Anopheles gambiae Y chromosome,” Proc. Natl. Acad. Sci. USA, vol. 111(21), pp. 7600-7605, May 12, 2014. [cited by applicant]
Byrne et al., “Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells,” Nucleic Acids Research, vol. 43(3), p. e21, 2014 (epub Nov. 20, 2014). [cited by applicant]
Chen et al., “High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases,” Nature Methods, vol. 8(9), pp. 753-755, 2011. (Jul. 17, 2011). [cited by applicant]
Choulika et al., “Induction of Homologous Recombination in Mammalian Chromosomes by Using the I-Scel System of [cited by applicant]
Cobb and Zhao, “Direct cloning of large genomic sequences,” Nature Biotechnology, 2012, vol. 30(5), pp. 405-406. [cited by applicant]
Cong et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems,” Science, vol. 339(6121), pp. 819-823 plus Supplemental Materials, Jan. 3, 2013. [cited by applicant]
Cui, et al., “Targeted integration in rat and mouse embryos with zinc-finger nucleases,” Nature Biotechnology, vol. 29, No. 1, 64-68 (Dec. 12, 2010). [cited by applicant]
Dechiara, T.M., et al., “VelociMouse: Fully ES Cell-Derived FO-Generation Mice Obtained from the Injection of ES Cells into Eight-Cell-Stage Embryos,” Jan. 1, 2009, Methods in Molecular Biology, 530(16): 311-324. [cited by applicant]
Donoho et al., “Analysis of Gene Targeting and Intrachromosomal Homologous Recombination Stimulated by Genomic Double-Strand Breaks in Mouse Embryonic Stem Cells,” Mol. Cell. Biol., vol. 18(7), pp. 4070-4078, 1998. [cited by applicant]
Doudna et al., “The new frontier of genome engineering with CRISPR-Cas9,” Science, vol. 346(6213), pp. 1258096-1-1258096-9, Nov. 28, 2014. [cited by applicant]
EP Application No. 14784879.0, Extended European Search Report mailed Sep. 19, 2016. [cited by applicant]
EP Application No. 19201384.5 Extended European Search Report mailed Feb. 19, 2020. [cited by applicant]
Evers et al., “CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes,” Nature Biotechnology, vol. 24(6), pp. 631-633, Apr. 25, 2016. [cited by applicant]
Fan et al., “107 Genetic Inactivation of the Sry Gene in Argali Wild and Romney Domestic Sheep with CRISPR/Cas Systems for Producing Sex-Reversed Female Animals,” Reproduction Fertility and Development, vol. 26(1), p. 1… [cited by applicant]
Flisikowska, et al., “Efficient Immunoglobulin Gene Disruption and Targeted Replacement in Rabbit Using Zinc Finger Nucleases,” Plos one, vol. 6 Issue 6 (Jun. 2011). [cited by applicant]
Frendewey, “VelociGene: Large-scale Modification of Rodent Genomes,” Wellcome Trust Advanced Course: Genetic Engineering of Mammalian Stem Cells, Feb. 20, 2015. [cited by applicant]
Frendewey, “VelociGene: Large-scale Modification of Rodent Genomes,” Wellcome Trust Advanced Course: Genetic Engineering of Mammalian Stem Cells, Mar. 13, 2014. [cited by applicant]
Fujii et al., “Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system,” Biochemical and Biophysical Research Communications, vol. 445(4), pp. 791-794 plus Supplementary Information, Jan. 31… [cited by applicant]
Gao et al., “DNA-guided genome editing using the Natronobacterium gregoryi Argonuate,” Nature Biotechnology, vol. 34(7), pp. 768-773, May 2, 2016. [cited by applicant]
Gennequin, et al., “CRISPR/Cas-induced double-strand breaks boost the frequency of gene replacements for humanizing the mouse Cnr2 gene,” Biochem. Biophys. Res. Commun., (2013), http://dx.doi.org/10.1016/j.bbrc.2013.10.… [cited by applicant]
Gibson, D., et al., “Enzymatic assembly of DNA molecules up to several hundred kilobases,” Nature Methods, 2009, vol. 6(5), pp. 343-345. [cited by applicant]
Gibson, Daniel G., “Enzymatic Assembly of Overlapping DNA Fragments,” Methods in Enzymology, 2011, vol. 498, pp. 349-361. [cited by applicant]
Gratz et al., “Genome Engineering of [cited by applicant]
Gratz et al., “Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in [cited by applicant]
Guo, et al., “Asymmetric DNA bending in the Cre-loxP site-specific recombination synapse,” Proc. Natl. Acad. Sci. U.S.A., 96(13):7143-7148, (Jun. 1999). [cited by applicant]
Houdebine, “Transgenic animal models in biomedical research,” Methods Mol. Biol. 360:163-202, (2007). [cited by applicant]
Jallepalli et al., “Securin is required for chromosomal stability in human cells,” Cell, vol. 105(4), pp. 445-457, May 18, 2001. [cited by applicant]
Jao et al., “Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system,” Proc. Natl. Acad. Sci. U.S.A., vol. 110(34), pp. 13904-13909 plus Supporting Information, Aug. 5, 2013. [cited by applicant]
Jasin, et al., “Repair of Strand Breaks by Homologous Recombination,” Cold Spring Harb. Perspect. Biol., vol. 5(11), p. a012740, Nov. 1, 2013. [cited by applicant]
Jiang, et al., “RNA-guided editing of bacterial genomes using CRISPR-Cas systems,” Nat. Biotechnol., 31(3):233-239, (2013). [cited by applicant]
Johnson, et al., “A rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta,” Plant Mol Biol, 82:207-221 (2013). [cited by applicant]
Kashimada et al., “Sry: the master switch in mammalian sex determination,” Development, vol. 137(23), pp. 3921-3930, Dec. 2, 2010. [cited by applicant]
Kato et al., “Production of Sry knockout mouse using TALEN via oocyte injection,” Scientific Reports, vol. 3, p. 3136, 2013 (published Nov. 5, 2013). [cited by applicant]
Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded cleavage,” Nature, vol. 533(7603), pp. 420-424, Apr. 20, 2016. [cited by applicant]
Konermann et al., “Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex,” Nature, vol. 517(7536), pp. 583-588, published online Dec. 10, 2014. [cited by applicant]
Kosicki et al., “Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements,” Nat. Biotechnol., 36(8): 765-771, (Jul. 16, 2018). [cited by applicant]
Kuijpers et al., “One-step assembly and targeted integration of multigene constructs assisted by the I-Scel meganuclease in [cited by applicant]
Kuno et al., “Generation of fertile and fecund F0 XY female mice from XY ES cells,” Transgenic Research, vol. 24(1), pp. 19-29, 2014 (epub Aug. 3, 2014). [cited by applicant]
Kuroiwa, et al., “Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle,” Nature Genetics, vol. 36, No. 7, (Jul. 2004). [cited by applicant]
Li et al., “Germline Competent Embryonic Stem Cells Derived from Rat Blastocysts,” Cell, vol. 135, pp. 1299-1310, 2008. [cited by applicant]
Li et al., “Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System,” PLoS One, vol. 9(8), p. e105779, Aug. 28, 2014. [cited by applicant]
Lin, S .- C., et al., “Strategies for gene disruption in Drosophila,” Cell & Bioscience (2014), vol. 4(1), p. 63. [cited by applicant]
Liu et al., “A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines,” BMC Biotechnol., vol. 12, p. 71, Oct. 9, 2012. [cited by applicant]
Lombardo, et al., “Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery,” Nature Biotechnology, 2007, vol. 25(11), pp. 1298-1306. [cited by applicant]
Macdonald, et al., “Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes,” PNAS, vol. 111, No. 14: 5147-5152, (Apr. 8, 2014). [cited by applicant]
Mali et al., “RNA-Guided Human Genome Engineering via Cas9,” Science, vol. 339(6121), pp. 823-826 plus Supplemental Materials, Jan. 3, 2013. [cited by applicant]
Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nature Biotechnology, vol. 31(9), pp. 833-838 plus Supplementary Information, Aug. … [cited by applicant]
Mali, et al., “Cas9 as a versatile tool for engineering biology,” Nature Methods, vol. 10(10), pp. 957-963, 2013. [cited by applicant]
Mashiko et al., “Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA,” Sci. Rep., vol. 3, p. 3355, Nov. 27, 2013. [cited by applicant]
Mashimo et al., “Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc finger nucleases,” PLoS One, vol. 5(1), p. e8870, 2010. [cited by applicant]
Morgens et al., “Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes,” Nature Biotechnology, vol. 34(6), pp. 634-636, May 9, 2016. [cited by applicant]
Musser, “Rodent,” Brittanica. Retrieved from the Internet May 31, 2016: http://www.brittanica.com/animal/rodent. [cited by applicant]
Narsinh, et al., “Gene Correction in Human Embryonic and Induced Pluripotent Stem Cells: Promise and Challenges Ahead”, Molecular Therapy, vol. 18, No. 6, pp. 1061-1063, (Jun. 2010). [cited by applicant]
Parikh et al., “Detailed Phenotypic and Molecular Analyses of Genetically Modified Mice Generated by CRISPR-Cas9-Mediated Editing,” PLoS One, vol. 10(1), p. e0116484, Jan. 14, 2015. [cited by applicant]
PCT International Preliminary Report on Patentability for application PCT/US2013/038165 mailed Oct. 28, 2014. [cited by applicant]
PCT International Preliminary Report on Patentability for application PCT/US2014/034412 mailed Oct. 30, 2015. [cited by applicant]
PCT International Preliminary Report on Patentability for application PCT/US2014/060788 mailed Jun. 23, 2016. [cited by applicant]
PCT International Search Report and Written Opinion of the International Searching Authority for application PCT/US2014/060788 mailed Jan. 26, 2015. [cited by applicant]
PCT International Search Report and Written Opinion of the International Searching Authority for application PCT/US2015/038001 mailed Feb. 25, 2016. [cited by applicant]
PCT International Search Report and Written Opinion of the International Searching Authority for application PCT/US2015/062023 mailed May 13, 2016. [cited by applicant]
PCT International Search Report and Written Opinion of the Internationalfor application PCT/US2015/066681 mailed Mar. 29, 2016. [cited by applicant]
PCT International Search Report for application PCT/US2015/034503 mailed Sep. 8, 2015. [cited by applicant]
PCT Written Opinion of the International Searching Authority for application PCT/US2015/034503 mailed Sep. 8, 2015. [cited by applicant]
PCT/US2013/038165 International Search Report and Written Opinion mailed Jul. 12, 2013. [cited by applicant]
PCT/US2014/034412 International Search Report and Written Opinion of the Searching Authority mailed Oct. 9, 2014. [cited by applicant]
PCT/US2015/038001 Invitation of Pay Additional Fees mailed Nov. 13, 2015. [cited by applicant]
PCT/US2015/062023 Invitation of Pay Additional Fees mailed Feb. 8, 2016. [cited by applicant]
Peng, Y., et al., “Making designer mutants in model organisms,” Development (2014), vol. 141, pp. 4042-4054. [cited by applicant]
Port et al., “Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in [cited by applicant]
Porteus, et al., “Gene targeting using zinc finger nucleases,” Nature23(8), pp. 967-973, 2005. [cited by applicant]
Poueymirou et al., “FO generation mice fully derived from gene-targeted embryonic stem cells allowing immediate analysis,” Nature Biotechnology, Epub Dec. 24, 2006, vol. 25(1):91-99. [cited by applicant]
Quinn et al., “A Site-Specific, Single-Copy Transgenesis Strategy to Identify 5′ Regulatory Sequences of the Mouse Testis-Determining Gene Sry,” PLoS One, vol. 9(4), p. e94813, Apr. 2014. [cited by applicant]
Ran et al., “Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity,” Cell, vol. 154, pp. 1380-1389, 2013. [cited by applicant]
Ran et al., “Genome engineering using the CRISPR-Cas9 system,” Nature Protocols, vol. 8(11), pp. 2281-2308, Oct. 24, 2013. [cited by applicant]
Ran et al., “In vivo genome editing using [cited by applicant]
Rong et al., “Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template,” Protein Cell 5(4):258-260, (2014). [cited by applicant]
Schwank, G., et al., “Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients,” Cell Stem Cell (2013), vol. 13, pp. 653-658. [cited by applicant]
Shan et al., “Targeted genome modification of crop plants using a CRISPR-Cas system,” Nature Biotechnology, vol. 31(8), pp. 686-688, Aug. 1, 2013. [cited by applicant]
Siao et al., “Single-step homozygous humanization induced by dual CRISPR/Cas9 cleavage,” Oct. 28, 2015. [cited by applicant]
Stemgent Product Specification Sheet, PD0325901, pp. 1-2 (2012). [cited by applicant]
Tong et al., “Generating gene knockout rats by homologous recombination in embryonic stem cells,” Nature Protocols, vol. 6(6), pp. 827-844, 2011 (epub May 26, 2011). [cited by applicant]
U.S. Appl. No. 13/870,280 Final Rejection mailed Oct. 15, 2015. [cited by applicant]
U.S. Appl. No. 13/870,280, Requirement for Restriction/Election mailed Jul. 22, 2014. [cited by applicant]
U.S. Appl. No. 14/254,715 Final Office Action mailed Nov. 30, 2015. [cited by applicant]
U.S. Appl. No. 14/254,715, Final Office Action mailed Sep. 19, 2016. [cited by applicant]
U.S. Appl. No. 14/254,715, Non-Final Office Action mailed Apr. 21, 2016. [cited by applicant]
U.S. Appl. No. 14/254,715, Non-Final Office Action mailed Aug. 12, 2015. [cited by applicant]
U.S. Appl. No. 14/314,866, Advisory Action mailed Aug. 12, 2015. [cited by applicant]
U.S. Appl. No. 14/314,866, Advisory Action mailed Aug. 15, 2016. [cited by applicant]
U.S. Appl. No. 14/314,866, Final Office Action mailed Apr. 26, 2016. [cited by applicant]
U.S. Appl. No. 14/314,866, Non-Final Office Action mailed Sep. 19, 2016. [cited by applicant]
U.S. Appl. No. 14/314,866, Non-Final Office Action mailed Dec. 26, 2014. [cited by applicant]
U.S. Appl. No. 14/314,866, Non-Final Office Action mailed Nov. 27, 2015. [cited by applicant]
U.S. Appl. No. 14/314,866, Requirement for Restriction/Election mailed Sep. 22, 2014. [cited by applicant]
U.S. Appl. No. 14/314,866, Final Office Action mailed Jun. 4, 2015. [cited by applicant]
U.S. Appl. No. 14/515,503, Non-Final Office Action mailed May 20, 2016. [cited by applicant]
U.S. Appl. No. 14/515,503, Notice of Allowance mailed Sep. 23, 2016. [cited by applicant]
U.S. Appl. No. 14/515,503, Requirement for Restriction/Election mailed Mar. 4, 2016. [cited by applicant]
U.S. Appl. No. 14/578,291, Non-Final Office Action mailed Mar. 10, 2015. [cited by applicant]
U.S. Appl. No. 14/578,291, Notice of Allowance mailed Aug. 26, 2015. [cited by applicant]
U.S. Appl. No. 14/731,914 , Requirement for Restriction/Election mailed Dec. 31, 2015. [cited by applicant]
U.S. Appl. No. 14/731,914, Non-Final Office Action mailed Jun. 17, 2016. [cited by applicant]
U.S. Appl. No. 14/751,807, Requirement for Restriction/Election mailed Aug. 26, 2016. [cited by applicant]
U.S. Appl. No. 14/926,773, Non-Final Office Action mailed May 6, 2016. [cited by applicant]
U.S. Appl. No. 14/926,773, Requirement for Restriction/Election mailed Feb. 16, 2016. [cited by applicant]
U.S. Appl. No. 14/928,180, Advisory Action mailed Aug. 22, 2016. [cited by applicant]
U.S. Appl. No. 14/928,180, Final Office Action mailed Jun. 6, 2016. [cited by applicant]
U.S. Appl. No. 14/974,623 Final Office Action mailed Dec. 14, 2018. [cited by applicant]
U.S. Appl. No. 14/974,623, Advisory Action mailed May 13, 2020. [cited by applicant]
U.S. Appl. No. 14/974,623, Advisory Action mailed Oct. 18, 2017. [cited by applicant]
U.S. Appl. No. 14/974,623, Final Office Action mailed Feb. 21, 2020. [cited by applicant]
U.S. Appl. No. 14/974,623, Final Office Action mailed Mar. 15, 2021. [cited by applicant]
U.S. Appl. No. 14/974,623, Final Office Action mailed Jun. 30, 2017. [cited by applicant]
U.S. Appl. No. 14/974,623, Non-Final Office Action mailed Apr. 6, 2018. [cited by applicant]
U.S. Appl. No. 14/974,623, Non-Final Office Action mailed Aug. 16, 2019. [cited by applicant]
U.S. Appl. No. 14/974,623, Non-Final Office Action mailed Oct. 2, 2020. [cited by applicant]
U.S. Appl. No. 14/974,623, Non-Final Office Action mailed Dec. 16, 2016. [cited by applicant]
U.S. Appl. No. 14/974,623, Notice of Allowance mailed Jan. 6, 2022. [cited by applicant]
U.S. Appl. No. 13/870,280, Advisory Action mailed Jan. 5, 2016. [cited by applicant]
U.S. Appl. No. 13/870,280, Non-Final Office Action mailed Mar. 13, 2015. [cited by applicant]
U.S. Appl. No. 14/254,715, Requirement for Restriction/Election mailed Jun. 4, 2015. [cited by applicant]
U.S. Appl. No. 14/928,180, Non-Final Office Action mailed Jan. 5, 2016. [cited by applicant]
Wang et al., “One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering,” Cell, vol. 153, pp. 910-918 plus supplemental materials, 2013. (published May 2013). [cited by applicant]
Wang et al., “TALEN-mediated editing of the mouse Y chromosome,” Nature Biotechnology, vol. 31(6), p. 530-532, 2013 (epub May 12, 2013). [cited by applicant]
Wen et al., “Completely ES Cell-Derived Mice Produced by Tetraploid Complementation Using Inner Cell Mass (ICM) Deficient Blastocysts,” PLoS One, vol. 9(4), e94730, Apr. 14, 2014. [cited by applicant]
Whitworth et al., “Use of the CRISPR/Cas9 System to Produce Genetically Engineered Pigs from In Vitro-Derived Oocytes and Embryos,” Biology of Reproduction, vol. 91(3), p. 78, Aug. 6, 2014. [cited by applicant]
Yoshimi et al., “Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR-Cas platform,” Nature Communications, vol. 5, p. 4240 plus Supplementary Information, Jun. 26, 201… [cited by applicant]
Yoshimi et al., “ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes,” Nat. Commun., vol. 7, p. 10431, Jan. 20, 2016. [cited by applicant]
Yu et al., “Highly Efficient Genome Modifications Mediated by CRISPR/Cas9 in [cited by applicant]
Zhang et al., “Biallelic targeting of expressed genes in mouse embryonic stem cells using the Cas9 system,” Methods, vol. 69(2), pp. 171-178, Jun. 12, 2014. [cited by applicant]
Zhou, H., et al., “Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice,” Nucleic Acids Research (2014), vol. 42(17), pp. 10903-10914. [cited by applicant]