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1. A highly efficient transgene knock-in technology in clinically relevant cell types

Author

Allen, Alexander G., Khan, Samia Q., Margulies, Carrie M., Viswanathan, Ramya, Lele, Swarali, Blaha, Laura, Scott, Sean N., Izzo, Kaitlyn M., Gerew, Alexandra, Pattali, Rithu, Cochran, Nadire R., Holland, Carl S., Zhao, Amy H., Sherman, Stephen E., Jaskolka, Michael C., Wu, Meng, Wilson, Aaron C., Sun, Xiaoqi, Ciulla, Dawn M., Zhang, Deric, Nelson, Jacqueline D., Zhang, Peisheng, Mazzucato, Patrizia, Huang, Yan, Giannoukos, Georgia, Marco, Eugenio, Nehil, Michael, Follit, John A., Chang, Kai-Hsin, Shearman, Mark S., Wilson, Christopher J., and Zuris, John A.

Published
2024
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3. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

Author

Komor, Alexis C, Kim, Yongjoo B, Packer, Michael S, Zuris, John A, and Liu, David R

Subjects
Biotechnology, Human Genome, Genetics, Good Health and Well Being, APOBEC-1 Deaminase, Animals, Apolipoprotein E4, Base Sequence, CRISPR-Associated Proteins, CRISPR-Cas Systems, Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats, Cytidine, Cytidine Deaminase, DNA, DNA Cleavage, DNA Repair, Deoxyribonuclease I, Genes, p53, Genetic Engineering, Genome, Humans, INDEL Mutation, Mice, Point Mutation, RNA, Guide, Kinetoplastida, Templates, Genetic, Uracil-DNA Glycosidase, Uridine, General Science & Technology
Abstract

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

Published
2016

4. Author Correction: AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

Author

Zhang, Liyang, Zuris, John A., Viswanathan, Ramya, Edelstein, Jasmine N., Turk, Rolf, Thommandru, Bernice, Rube, H. Tomas, Glenn, Steve E., Collingwood, Michael A., Bode, Nicole M., Beaudoin, Sarah F., Lele, Swarali, Scott, Sean N., Wasko, Kevin M., Sexton, Steven, Borges, Christopher M., Schubert, Mollie S., Kurgan, Gavin L., McNeill, Matthew S., Fernandez, Cecilia A., Myer, Vic E., Morgan, Richard A., Behlke, Mark A., and Vakulskas, Christopher A.

Published
2021
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5. AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

Author

Zhang, Liyang, Zuris, John A., Viswanathan, Ramya, Edelstein, Jasmine N., Turk, Rolf, Thommandru, Bernice, Rube, H. Tomas, Glenn, Steve E., Collingwood, Michael A., Bode, Nicole M., Beaudoin, Sarah F., Lele, Swarali, Scott, Sean N., Wasko, Kevin M., Sexton, Steven, Borges, Christopher M., Schubert, Mollie S., Kurgan, Gavin L., McNeill, Matthew S., Fernandez, Cecilia A., Myer, Vic E., Morgan, Richard A., Behlke, Mark A., and Vakulskas, Christopher A.

Published
2021
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6. A highly efficient transgene knock-in technology in clinically relevant cell types

Author

Allen, Alexander G., primary, Khan, Samia Q., additional, Margulies, Carrie M., additional, Viswanathan, Ramya, additional, Lele, Swarali, additional, Blaha, Laura, additional, Scott, Sean N., additional, Izzo, Kaitlyn M., additional, Gerew, Alexandra, additional, Pattali, Rithu, additional, Cochran, Nadire R., additional, Holland, Carl S., additional, Zhao, Amy H., additional, Sherman, Stephen E., additional, Jaskolka, Michael C., additional, Wu, Meng, additional, Wilson, Aaron C., additional, Sun, Xiaoqi, additional, Ciulla, Dawn M., additional, Zhang, Deric, additional, Nelson, Jacqueline D., additional, Zhang, Peisheng, additional, Mazzucato, Patrizia, additional, Huang, Yan, additional, Giannoukos, Georgia, additional, Marco, Eugenio, additional, Nehil, Michael, additional, Follit, John A., additional, Chang, Kai-Hsin, additional, Shearman, Mark S., additional, Wilson, Christopher J., additional, and Zuris, John A., additional

Published
2023
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9. Treatment of monogenic and digenic dominant genetic hearing loss by CRISPR-Cas9 ribonucleoprotein delivery in vivo

Author

Alvarez, Veronica Lamas, primary, Tao, Yong, additional, Li, Yiran, additional, Du, Wan, additional, Whittaker, Madelynn, additional, Zuris, John, additional, Thompson, David, additional, Zhu, Wenliang, additional, Rameshbabu, Arun Prabhu, additional, Shu, Yilai, additional, Gao, Xue, additional, Hu, Johnny, additional, Kong, Wei-Jia, additional, Liu, Xueazhong, additional, Wu, Hao, additional, Kleinstiver, Benjamin, additional, Liu, David, additional, and Chen, Zheng-Yi, additional

Published
2022
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16. Unraveling the biological role and emerging capabilities of a novel class of Type 2 diabetes drug targets

Author

Zuris, John A.

Subjects
UCSD Dissertations, Academic Chemistry. (Discipline)
Abstract

MitoNEET (mNT) is an outer mitochondrial membrane iron- sulfur (FeS) protein and a target of the thiazolidinedione (TZD) class of anti-diabetes drugs. The [2Fe-2S] cluster in mNT is ligated by a rare 3-Cys-1-His coordination. As FeS proteins usually function in electron transfer (redox) or in transfer of their clusters to apo-acceptor proteins, we investigated whether the TZDs affect the protein's redox potential (EM,7). We found that TZD binding negatively shifts EM, 7 by ̃100 mV, whereas in the cluster -coordinating His87 to Cys mutant (H87C) this effect is not observed. This suggests that His87 is critical to TZD communication with the [2Fe-2S] cluster of mNT. We further investigated the importance of residues near the cluster and discovered that mNT could tolerate an array of mutations that modified EM, 7 over a range of ∼700 mV. This is the largest range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to -300 mV). Therefore, mNT is potentially useful for both physiological redox studies and industrial applications as a stable, water-soluble, redox agent. We investigated whether mNT functions as a cluster transfer protein and if TZDs affect transfer. We observed facile [2Fe-2S] cluster transfer between oxidized mNT and apo-ferredoxin (a-Fd) both in vitro and with a mitochondrial iron detection assay in cells. The H87C mutant inhibits transfer of the [2Fe-2S] clusters both in vitro and in cells. Importantly, TZDs inhibit iron transfer from mNT to mitochondria. This finding is interesting in light of the role of iron overload in diabetes. Finally, we show that NADPH shifts EM, 7 negatively and inhibits cluster transfer in a manner similar to the TZDs, The most critical cellular function of NADPH is in the maintenance of a pool of reducing equivalents, which is essential to counteract oxidative damage. Taken together, our findings suggest a likely role for mNT in [2Fe-2S] and/or iron transfer to acceptor proteins and support the idea that pioglitazone's anti- diabetic mode of action may, in part, be to inhibit transfer of mNT's [2Fe-2S] cluster and protect cells from iron-overload

Published
2012

17. 145 Preclinical development of EDIT-201, a multiplexed CRISPR-Cas12a gene edited healthy donor derived NK cells demonstrating improved persistence and resistance to the tumor microenvironment

Author

Wong, Karrie, primary, Sexton, Steven, additional, Donahue, Kelly, additional, Antony, Lincy Prem, additional, Wasko, Kevin, additional, Nasser, Jared, additional, Leary, Glenn, additional, Pfautz, Amanda, additional, Porth, Owen, additional, Pierce, William, additional, Sousa, Patricia, additional, Scott, Sean, additional, Wilson, Aaron, additional, Chang, Kai-Hsin, additional, Zuris, John, additional, Wilson, Christopher, additional, Morgan, Richard, additional, and Borges, Christopher, additional

Published
2020
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19. EDIT-301: An Experimental Autologous Cell Therapy Comprising Cas12a-RNP Modified mPB-CD34+ Cells for the Potential Treatment of SCD

Author

De Dreuzy, Edouard, primary, Heath, Jack, additional, Zuris, John A, additional, Sousa, Patricia, additional, Viswanathan, Ramya, additional, Scott, Sean, additional, Da Silva, Jen, additional, Ta, Terence, additional, Capehart, Stacy, additional, Wang, Tongyao, additional, Fernandez, Cecilia, additional, Myer, Vic E, additional, Albright, Charles F, additional, Wilson, Christopher J, additional, Teixeira, Sandra, additional, and Chang, Kai-Hsin, additional

Published
2019
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20. Comparative Studies Reveal Robust HbF Induction By Editing of HBG1/2 Promoters or BCL11A Erythroid-Enhancer in Human CD34+ Cells but That BCL11A Erythroid-Enhancer Editing Is Associated with Selective Reduction in Erythroid Lineage Reconstitution in a Xenotransplantation Model

Author

Chang, Kai-Hsin, primary, Sanchez, Minerva, additional, Heath, Jack, additional, deDreuzy, Edouard, additional, Haskett, Scott, additional, Vogelaar, Abigail, additional, Gogi, Kiran, additional, Da Silva, Jen, additional, Wang, Tongyao, additional, Sadowski, Andrew, additional, Gotta, Gregory, additional, Siwak, Jamaica, additional, Viswanathan, Ramya, additional, Loveluck, Katherine, additional, Chao, Hoson, additional, Tillotson, Eric, additional, Chalishazar, Aditi, additional, Dass, Abhishek, additional, Ta, Frederick, additional, Brennan, Emily, additional, Tabbaa, Diana, additional, Marco, Eugenio, additional, Zuris, John A, additional, Reyon, Deepak, additional, Isik, Meltem, additional, Friedland, Ari, additional, Ta, Terence, additional, Harbinski, Fred, additional, Giannoukos, Georgia, additional, Teixeira, Sandra, additional, Wilson, Christopher J, additional, Albright, Charlie, additional, and Jiang, Haiyan, additional

Published
2018
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21. Engineering the redox potential over a wide range within a new class of FeS proteins

Author

Zuris, John A., Halim, Danny A., Conlan, Andrea R., Abresch, Edward C., Nechushtai, Rachel, Paddock, Mark L., and Jennings, Patricia A.

Subjects
Gene mutations -- Analysis, Iron compounds -- Structure, Iron compounds -- Chemical properties, Mitochondria -- Physiological aspects, Oxidation-reduction reaction -- Analysis, Protein binding -- Analysis, Chemistry
Abstract

A newly discovered MitoNEET mitochondrial protein and a target of the TZD class of antidiabetes drugs, which are homodimeric with each protomer binding a [2Fe-2S] center through a rare 3-Cys and 1-His coordination geometry is reported. The ability of mitoNEET protein to tolerate an array of mutations that modified the redox potential [E.sub.M] of the [2Fe-2S] center over a range of ~700 mV could find potential use for both physiological studies and industrial applications as a stable, water-soluble, redox agent.

Published
2010

22. Redox characterization of the FeS protein MitoNEET and impact of thiazolidinedione drug binding

Author

Bak, Daniel W., Zuris, John A., Paddock, Mark L., Jennings, Patricia A., and Elliott, Sean J.

Subjects
Oxidation-reduction reaction -- Analysis, Protein binding -- Analysis, Voltammetry -- Usage, Mitochondrial DNA -- Chemical properties, Gene mutations -- Analysis, Histidine -- Chemical properties, Biological sciences, Chemistry
Abstract

The protein film voltammetry (PFV) was used to probe the redox properties of mitoNEET and demonstrate the direct impact of thiazolidinedione (TZD) drug binding upon the redox chemistry of the FeS cluster. A His87Cys mutant which negated the ability of TZDs to affect the midpoint potential suggested a model of drug binding in which His87 was critical to communication with the FeS center of mitoNEET.

Published
2009

26. Nutrient-Deprivation Autophagy Factor-1 (NAF-1): Biochemical Properties of a Novel Cellular Target for Anti-Diabetic Drugs

Author

Tamir, Sagi, primary, Zuris, John A., additional, Agranat, Lily, additional, Lipper, Colin H., additional, Conlan, Andrea R., additional, Michaeli, Dorit, additional, Harir, Yael, additional, Paddock, Mark L., additional, Mittler, Ron, additional, Cabantchik, Zvi Ioav, additional, Jennings, Patricia A., additional, and Nechushtai, Rachel, additional

Published
2013
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27. Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.

Author

Ming Wang, Zuris, John A., Fantao Meng, Rees, Holly, Shuo Sun, Pu Deng, Yong Han, Xue Gao, Pouli, Dimitra, Qi Wu, Georgakoudi, Irene, Liu, David R., and Qiaobing Xu

Subjects
*PROTEIN analysis, *LIPIDS, *NANOPARTICLES analysis, *ENDOSOMES, *GENETIC recombination
Abstract

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing. [ABSTRACT FROM AUTHOR]

Published
2016
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28. Allosteric control in a metalloprotein dramatically alters function

Author

Baxter, Elizabeth Leigh, primary, Zuris, John A., additional, Wang, Charles, additional, Vo, Phu Luong T., additional, Axelrod, Herbert L., additional, Cohen, Aina E., additional, Paddock, Mark L., additional, Nechushtai, Rachel, additional, Onuchic, Jose N., additional, and Jennings, Patricia A., additional

Published
2012
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37. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Author

Zuris, John A, Thompson, David B, Shu, Yilai, Guilinger, John P, Bessen, Jeffrey L, Hu, Johnny H, Maeder, Morgan L, Joung, J Keith, Chen, Zheng-Yi, and Liu, David R

Subjects
*GENOME editing, *GENE transfection, *PROTEIN genetics, *LIPIDS, *RNA, *RECOMBINASES, *IN vitro studies
Abstract

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells. [ABSTRACT FROM AUTHOR]

Published
2015
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38. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

Author

Komor, Alexis C., Kim, Yongjoo B., Packer, Michael S., Zuris, John A., and Liu, David R.

Subjects
Genome editing, protein engineering, CRISPR, Cas9, genetic diseases, single-nucleotide polymorphism
Abstract

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction.1,2 Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus from the cellular response to dsDNA breaks.1,2 Here we report the development of base editing, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting “base editors” convert cytidines within a window of approximately five nucleotides (nt), and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor (UGI), and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favor desired base-editing outcomes, resulting in permanent correction of ∼15-75% of total cellular DNA with minimal (typically ≤ 1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

Published
2016
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39. Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo

Author

Zuris, John A., Thompson, David B., Shu, Yilai, Guilinger, John P., Bessen, Jeffrey L., Hu, Johnny H., Maeder, Morgan L., Joung, J. Keith, Chen, Zheng-Yi, and Liu, David R.

Abstract

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape, and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Published
2014
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40. Comparative Studies Reveal Robust HbF Induction By Editing of HBG1/2Promoters or BCL11AErythroid-Enhancer in Human CD34+ Cells but That BCL11AErythroid-Enhancer Editing Is Associated with Selective Reduction in Erythroid Lineage Reconstitution in a Xenotransplantation Model

Author

Chang, Kai-Hsin, Sanchez, Minerva, Heath, Jack, deDreuzy, Edouard, Haskett, Scott, Vogelaar, Abigail, Gogi, Kiran, Da Silva, Jen, Wang, Tongyao, Sadowski, Andrew, Gotta, Gregory, Siwak, Jamaica, Viswanathan, Ramya, Loveluck, Katherine, Chao, Hoson, Tillotson, Eric, Chalishazar, Aditi, Dass, Abhishek, Ta, Frederick, Brennan, Emily, Tabbaa, Diana, Marco, Eugenio, Zuris, John A, Reyon, Deepak, Isik, Meltem, Friedland, Ari, Ta, Terence, Harbinski, Fred, Giannoukos, Georgia, Teixeira, Sandra, Wilson, Christopher J, Albright, Charlie, and Jiang, Haiyan

Published
2018
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41. Expanding CRISPR Genome Editing Strategies in Hematopoietic Stem and Progenitor Cells for the Treatment of Hematologic Diseases

Author

deDreuzy, Edouard, Chalishazar, Aditi, Heath, Jack, Margulies, Carrie M, Labella, John, Viswanathan, Ramya, Loveluck, Katherine, Chang, KaiHsin, Jayaram, Hariharan, Zuris, John A, Myer, Vic E, Albright, Charlie, Cotta-Ramusino, Cecilia, and Gori, Jennifer L

Abstract

We have previously shown that CRISPR/Cas9 mediates high levels of gene editing in adult human CD34+hematopoietic stem and progenitor cells (HSPCs). We now demonstrated that high levels of gene editing can also be achieved in HSPCs using Cpf1. In addition to NHEJ-mediated gene editing, we explored targeted integration at the ß-hemoglobin gene (HBB) with Cas9, toward gene correction for sickle cell disease (SCD). For targeted integration strategies at HBB, we designed AAV6 DNA donor repair templates flanked by either DNA sequences homologous to the cut site in the genomic location of the SCD mutation or DNA sequences non-homologous to the human genome. To evaluate targeted integration in HSPCs, adult mobilized peripheral blood (mPB) CD34+cells were electroporated with Cas9 ribonucleoprotein (RNP) and were then transduced with AAV6 containing DNA repair templates with or without homology arms. We analyzed targeted integration at the HBBtarget site by digital droplet PCR (ddPCR) analysis 7 days after transduction of mPB CD34+cells. The ddPCR results show ~30% targeted integration in HBBin mPB CD34+cells with no impact on cell viability. DNA sequencing analysis supported the ddPCR results indicating that perfect homology directed repair-driven integration of the DNA donor template was the preferential repair mechanism. Less than 1% of targeted integration was detected by ddPCR in mPB CD34+cells transduced with AAV6 DNA repair template that lacked homology arms, suggesting that the mechanism of integration is homology repair dependent. These integrations were maintained in the Glycophorin A+erythroid progeny of genome-edited mPB CD34+cells, paving the way toward gene correction for SCD. The combination of CRISPR nucleases (Cpf1, Cas9) and AAV6 DNA repair templates form a powerful genome editing platform that enables broader applications for the treatment of hematologic diseases beyond hemoglobinopathies.

Published
2017
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