Publications by authors named "Thomas Gaj"

42 Publications

Targeted gene silencing in the nervous system with CRISPR-Cas13.

Sci Adv 2022 01 19;8(3):eabk2485. Epub 2022 Jan 19.

Department of Bioengineering, University of Illinois, Urbana, IL 61801, USA.

Cas13 nucleases are a class of programmable RNA-targeting CRISPR effector proteins that are capable of silencing target gene expression in mammalian cells. Here, we demonstrate that RfxCas13d, a Cas13 ortholog with favorable characteristics to other family members, can be delivered to the mouse spinal cord and brain to silence neurodegeneration-associated genes. Intrathecally delivering an adeno-associated virus vector encoding an RfxCas13d variant programmed to target superoxide dismutase 1 (SOD1), a protein whose mutation can cause amyotrophic lateral sclerosis, reduced SOD1 mRNA and protein in the spinal cord by >50% and improved outcomes in a mouse model of the disorder. We further show that intrastriatally delivering an RfxCas13d variant programmed to target huntingtin (HTT), a protein whose mutation is causative for Huntington’s disease, led to a ~50% reduction in HTT protein in the mouse brain. Our results establish RfxCas13d as a versatile platform for knocking down gene expression in the nervous system.
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http://dx.doi.org/10.1126/sciadv.abk2485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8769545PMC
January 2022

Next-Generation CRISPR Technologies and Their Applications in Gene and Cell Therapy.

Trends Biotechnol 2021 07;39(7):692-705

Department of Bioengineering, University of Illinois, Urbana, IL 61801, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, IL 61801, USA. Electronic address:

The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) nucleases has transformed biotechnology by providing an easy, efficient, and versatile platform for editing DNA. However, traditional CRISPR-based technologies initiate editing by activating DNA double-strand break (DSB) repair pathways, which can cause adverse effects in cells and restrict certain therapeutic applications of the technology. To this end, several new CRISPR-based modalities have been developed that are capable of catalyzing editing without the requirement for a DSB. Here, we review three of these technologies: base editors, prime editors, and RNA-targeting CRISPR-associated protein (Cas)13 effectors. We discuss their strengths compared to traditional gene-modifying systems, we highlight their emerging therapeutic applications, and we examine challenges facing their safe and effective clinical implementation.
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http://dx.doi.org/10.1016/j.tibtech.2020.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8166939PMC
July 2021

Treatment of a Mouse Model of ALS by In Vivo Base Editing.

Mol Ther 2020 04 14;28(4):1177-1189. Epub 2020 Jan 14.

Department of Bioengineering, University of Illinois, Urbana, IL 61801, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, IL 61801, USA. Electronic address:

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal disorder that can be caused by mutations in the superoxide dismutase 1 (SOD1) gene. Although ALS is currently incurable, CRISPR base editors hold the potential to treat the disease through their ability to create nonsense mutations that can permanently disable the expression of the mutant SOD1 gene. However, the restrictive carrying capacity of adeno-associated virus (AAV) vectors has limited their therapeutic application. In this study, we establish an intein-mediated trans-splicing system that enables in vivo delivery of cytidine base editors (CBEs) consisting of the widely used Cas9 protein from Streptococcus pyogenes. We show that intrathecal injection of dual AAV particles encoding a split-intein CBE engineered to trans-splice and introduce a nonsense-coding substitution into a mutant SOD1 gene prolonged survival and markedly slowed the progression of disease in the G93A-SOD1 mouse model of ALS. Adult animals treated by this split-intein CRISPR base editor had a reduced rate of muscle atrophy, decreased muscle denervation, improved neuromuscular function, and up to 40% fewer SOD1 immunoreactive inclusions at end-stage mice compared to control mice. This work expands the capabilities of single-base editors and demonstrates their potential for gene therapy.
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http://dx.doi.org/10.1016/j.ymthe.2020.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132599PMC
April 2020

CRISPR-Cas9-Mediated Genome Editing Increases Lifespan and Improves Motor Deficits in a Huntington's Disease Mouse Model.

Mol Ther Nucleic Acids 2019 Sep 26;17:829-839. Epub 2019 Jul 26.

Department of Bioengineering, University of Illinois, Urbana, IL, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, IL, USA. Electronic address:

Huntington's disease (HD) is a currently incurable and, ultimately, fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion within exon 1 of the huntingtin (HTT) gene, which results in the production of a mutant protein that forms inclusions and selectively destroys neurons in the striatum and other adjacent structures. The RNA-guided Cas9 endonuclease from CRISPR-Cas9 systems is a versatile technology for inducing DNA double-strand breaks that can stimulate the introduction of frameshift-inducing mutations and permanently disable mutant gene function. Here, we show that the Cas9 nuclease from Staphylococcus aureus, a small Cas9 ortholog that can be packaged alongside a single guide RNA into a single adeno-associated virus (AAV) vector, can be used to disrupt the expression of the mutant HTT gene in the R6/2 mouse model of HD following its in vivo delivery to the striatum. Specifically, we found that CRISPR-Cas9-mediated disruption of the mutant HTT gene resulted in a ∼50% decrease in neuronal inclusions and significantly improved lifespan and certain motor deficits. These results thus illustrate the potential for CRISPR-Cas9 technology to treat HD and other autosomal dominant neurodegenerative disorders caused by a trinucleotide repeat expansion via in vivo genome editing.
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http://dx.doi.org/10.1016/j.omtn.2019.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717077PMC
September 2019

The continuously evolving CRISPR barcoding toolbox.

Genome Biol 2018 09 25;19(1):143. Epub 2018 Sep 25.

Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.

Two articles recently described the development of CRISPR technologies that have the potential to fundamentally transform the barcoding and tracing of mammalian cells.
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http://dx.doi.org/10.1186/s13059-018-1541-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6154929PMC
September 2018

Manufacturing and Delivering Genome-Editing Proteins.

Methods Mol Biol 2018 ;1867:253-273

Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Genome-editing technologies have revolutionized the biomedical sciences by providing researchers with the ability to quickly and efficiently modify genes. While programmable nucleases can be introduced into cells using a variety of techniques, their delivery as purified proteins is an effective approach for limiting off-target effects. Here, we describe step-by-step procedures for manufacturing and delivering genome-modifying proteins-including Cas9 ribonucleoproteins (RNPs) and TALE and zinc-finger nucleases-into mammalian cells. Protocols for combining Cas9 RNP with naturally recombinogenic adeno-associated virus (AAV) donor vectors for the seamless insertion of transgenes by homology-directed genome editing are also provided.
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http://dx.doi.org/10.1007/978-1-4939-8799-3_19DOI Listing
July 2019

hPSC-Derived Striatal Cells Generated Using a Scalable 3D Hydrogel Promote Recovery in a Huntington Disease Mouse Model.

Stem Cell Reports 2018 05 5;10(5):1481-1491. Epub 2018 Apr 5.

Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; The Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address:

Huntington disease (HD) is an inherited, progressive neurological disorder characterized by degenerating striatal medium spiny neurons (MSNs). One promising approach for treating HD is cell replacement therapy, where lost cells are replaced by MSN progenitors derived from human pluripotent stem cells (hPSCs). While there has been remarkable progress in generating hPSC-derived MSNs, current production methods rely on two-dimensional culture systems that can include poorly defined components, limit scalability, and yield differing preclinical results. To facilitate clinical translation, here, we generated striatal progenitors from hPSCs within a fully defined and scalable PNIPAAm-PEG three-dimensional (3D) hydrogel. Transplantation of 3D-derived striatal progenitors into a transgenic mouse model of HD slowed disease progression, improved motor coordination, and increased survival. In addition, the transplanted cells developed an MSN-like phenotype and formed synaptic connections with host cells. Our results illustrate the potential of scalable 3D biomaterials for generating striatal progenitors for HD cell therapy.
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http://dx.doi.org/10.1016/j.stemcr.2018.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995679PMC
May 2018

Innovations in CRISPR technology.

Curr Opin Biotechnol 2018 08 5;52:95-101. Epub 2018 Apr 5.

Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA. Electronic address:

CRISPR-Cas9 is a versatile tool for genome engineering that has revolutionized biotechnology and is poised to impact medicine. Recent advances in the identification of unique CRISPR systems, as well as the re-engineering of the Cas9 protein for expanded function, has enabled the diversification of the CRISPR genome engineering toolbox. In this review, we highlight these innovations and discuss how advances in CRISPR technology can lead to breakthroughs in the field of gene therapy.
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http://dx.doi.org/10.1016/j.copbio.2018.03.007DOI Listing
August 2018

A Hypothalamic Switch for REM and Non-REM Sleep.

Neuron 2018 03 22;97(5):1168-1176.e4. Epub 2018 Feb 22.

Division of Neurobiology, Department of Molecular and Cell Biology, Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA. Electronic address:

Rapid eye movement (REM) and non-REM (NREM) sleep are controlled by specific neuronal circuits. Here we show that galanin-expressing GABAergic neurons in the dorsomedial hypothalamus (DMH) comprise separate subpopulations with opposing effects on REM versus NREM sleep. Microendoscopic calcium imaging revealed diverse sleep-wake activity of DMH GABAergic neurons, but the galanin-expressing subset falls into two distinct groups, either selectively activated (REM-on) or suppressed (REM-off) during REM sleep. Retrogradely labeled, preoptic area (POA)-projecting galaninergic neurons are REM-off, whereas the raphe pallidus (RPA)-projecting neurons are primarily REM-on. Bidirectional optogenetic manipulations showed that the POA-projecting neurons promote NREM sleep and suppress REM sleep, while the RPA-projecting neurons have the opposite effects. Thus, REM/NREM switch is regulated antagonistically by DMH galaninergic neurons with intermingled cell bodies but distinct axon projections.
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http://dx.doi.org/10.1016/j.neuron.2018.02.005DOI Listing
March 2018

In vivo genome editing improves motor function and extends survival in a mouse model of ALS.

Sci Adv 2017 12 20;3(12):eaar3952. Epub 2017 Dec 20.

Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord and brain. In particular, autosomal dominant mutations in the superoxide dismutase 1 (SOD1) gene are responsible for ~20% of all familial ALS cases. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas9) genome editing system holds the potential to treat autosomal dominant disorders by facilitating the introduction of frameshift-induced mutations that can disable mutant gene function. We demonstrate that CRISPR-Cas9 can be harnessed to disrupt mutant SOD1 expression in the G93A-SOD1 mouse model of ALS following in vivo delivery using an adeno-associated virus vector. Genome editing reduced mutant SOD1 protein by >2.5-fold in the lumbar and thoracic spinal cord, resulting in improved motor function and reduced muscle atrophy. Crucially, ALS mice treated by CRISPR-mediated genome editing had ~50% more motor neurons at end stage and displayed a ~37% delay in disease onset and a ~25% increase in survival compared to control animals. Thus, this study illustrates the potential for CRISPR-Cas9 to treat SOD1-linked forms of ALS and other central nervous system disorders caused by autosomal dominant mutations.
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http://dx.doi.org/10.1126/sciadv.aar3952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738228PMC
December 2017

Defined and Scalable Differentiation of Human Oligodendrocyte Precursors from Pluripotent Stem Cells in a 3D Culture System.

Stem Cell Reports 2017 06 25;8(6):1770-1783. Epub 2017 May 25.

Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720-1762, USA; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720-1462, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3370, USA; The Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720-3370, USA. Electronic address:

Oligodendrocyte precursor cells (OPCs) offer considerable potential for the treatment of demyelinating diseases and injuries of the CNS. However, generating large quantities of high-quality OPCs remains a substantial challenge that impedes their therapeutic application. Here, we show that OPCs can be generated from human pluripotent stem cells (hPSCs) in a three-dimensional (3D), scalable, and fully defined thermoresponsive biomaterial system. We used CRISPR/Cas9 to create a NKX2.2-EGFP human embryonic stem cell reporter line that enabled fine-tuning of early OPC specification and identification of conditions that markedly increased the number of OLIG2 and NKX2.2 cells generated from hPSCs. Transplantation of 50-day-old OPCs into the brains of NOD/SCID mice revealed that progenitors generated in 3D without cell selection or purification subsequently engrafted, migrated, and matured into myelinating oligodendrocytes in vivo. These results demonstrate the potential of harnessing lineage reporter lines to develop 3D platforms for rapid and large-scale production of OPCs.
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http://dx.doi.org/10.1016/j.stemcr.2017.04.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470111PMC
June 2017

Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.

Nucleic Acids Res 2017 Jun;45(11):e98

Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.

Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide. However, combining RNPs with transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homology-directed genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing.
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http://dx.doi.org/10.1093/nar/gkx154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499784PMC
June 2017

Genome-Editing Technologies: Principles and Applications.

Cold Spring Harb Perspect Biol 2016 Dec 1;8(12). Epub 2016 Dec 1.

Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China.

Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.
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http://dx.doi.org/10.1101/cshperspect.a023754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131771PMC
December 2016

Adeno-Associated Virus-Mediated Delivery of CRISPR-Cas Systems for Genome Engineering in Mammalian Cells.

Cold Spring Harb Protoc 2016 11 1;2016(11). Epub 2016 Nov 1.

Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720.

The CRISPR-Cas9 system has emerged as a highly versatile platform for introducing targeted genome modifications into mammalian cells and model organisms. However, fully capitalizing on the therapeutic potential for this system requires its safe and efficient delivery into relevant cell types. Adeno-associated virus (AAV) vectors are a clinically promising class of engineered gene-delivery vehicles capable of safely infecting a broad range of dividing and nondividing cell types, while also serving as a highly effective donor template for homology-directed repair. Together, CRISPR-Cas9 and AAV technologies have the potential to accelerate both basic research and clinical applications of genome engineering. Here, we present a step-by-step protocol for AAV-mediated delivery of CRISPR-Cas systems into mammalian cells. Procedures are given for the preparation of high-titer virus capable of achieving a diverse range of genetic modifications, including gene knockout and integration.
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http://dx.doi.org/10.1101/pdb.prot086868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850213PMC
November 2016

A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons.

Neuron 2016 Oct 6;92(2):372-382. Epub 2016 Oct 6.

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA. Electronic address:

Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV), a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.neuron.2016.09.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5872824PMC
October 2016

Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors.

PLoS One 2016 2;11(3):e0150037. Epub 2016 Mar 2.

Research Institute for Medicines (iMed ULisboa), Faculdadede Farmácia, Universidade de Lisboa, Lisboa, Portugal.

The presence of replication-competent HIV-1 -which resides mainly in resting CD4+ T cells--is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150037PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4774903PMC
July 2016

CRISPR-mediated Activation of Latent HIV-1 Expression.

Mol Ther 2016 Mar 26;24(3):499-507. Epub 2015 Nov 26.

Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, California, USA.

Complete eradication of HIV-1 infection is impeded by the existence of cells that harbor chromosomally integrated but transcriptionally inactive provirus. These cells can persist for years without producing viral progeny, rendering them refractory to immune surveillance and antiretroviral therapy and providing a permanent reservoir for the stochastic reactivation and reseeding of HIV-1. Strategies for purging this latent reservoir are thus needed to eradicate infection. Here, we show that engineered transcriptional activation systems based on CRISPR/Cas9 can be harnessed to activate viral gene expression in cell line models of HIV-1 latency. We further demonstrate that complementing Cas9 activators with latency-reversing compounds can enhance latent HIV-1 transcription and that epigenome modulation using CRISPR-based acetyltransferases can also promote viral gene activation. Collectively, these results demonstrate that CRISPR systems are potentially effective tools for inducing latent HIV-1 expression and that their use, in combination with antiretroviral therapy, could lead to improved therapies for HIV-1 infection.
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http://dx.doi.org/10.1038/mt.2015.213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786916PMC
March 2016

Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells.

Nat Protoc 2015 Nov 22;10(11):1842-59. Epub 2015 Oct 22.

The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.

Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity. Once they are constructed, nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d.
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http://dx.doi.org/10.1038/nprot.2015.117DOI Listing
November 2015

Redesigning Recombinase Specificity for Safe Harbor Sites in the Human Genome.

PLoS One 2015 28;10(9):e0139123. Epub 2015 Sep 28.

The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037, United States of America; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037, United States of America; Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA, 92037, United States of America.

Site-specific recombinases (SSRs) are valuable tools for genetic engineering due to their ability to manipulate DNA in a highly specific manner. Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases. While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme. In order to further expand the targeted recombinase repertoire, we used a genetic screen to isolate enhanced mutants of the Bin and Tn21 recombinases that recognize target sites outside the scope of other engineered recombinases. We determined the specific base requirements for recombination by these enzymes and demonstrate their potential for genome engineering by selecting for variants capable of specifically recombining target sites present in the human CCR5 gene and the AAVS1 safe harbor locus. Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139123PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587366PMC
May 2016

Genome Engineering Using Adeno-associated Virus: Basic and Clinical Research Applications.

Mol Ther 2016 Mar 16;24(3):458-64. Epub 2015 Sep 16.

Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA.

In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination in mammalian cells at high efficiencies. This process--referred to as AAV-mediated gene targeting--has enabled the introduction of a diverse array of genomic modifications both in vitro and in vivo. With the recent emergence of targeted nucleases, AAV-mediated genome engineering is poised for clinical translation. Here, we review key properties of AAV vectors that underscore its unique utility in genome editing. We highlight the broad range of genome engineering applications facilitated by this technology and discuss the strong potential for unifying AAV with targeted nucleases for next-generation gene therapy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786909PMC
http://dx.doi.org/10.1038/mt.2015.151DOI Listing
March 2016

Direct protein delivery to mammalian cells using cell-permeable Cys2-His2 zinc-finger domains.

Authors:
Thomas Gaj Jia Liu

J Vis Exp 2015 Mar 25(97). Epub 2015 Mar 25.

Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute; Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.
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http://dx.doi.org/10.3791/52814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4401377PMC
March 2015

Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering.

Mol Ther Nucleic Acids 2015 Mar 10;4:e232. Epub 2015 Mar 10.

1] The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA [2] Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA [3] Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA [4] Deceased.

Safe, efficient, and broadly applicable methods for delivering site-specific nucleases into cells are needed in order for targeted genome editing to reach its full potential for basic research and medicine. We previously reported that zinc-finger nuclease (ZFN) proteins have the innate capacity to cross cell membranes and induce genome modification via their direct application to human cells. Here, we show that incorporation of tandem nuclear localization signal (NLS) repeats into the ZFN protein backbone enhances cell permeability nearly 13-fold and that single administration of multi-NLS ZFN proteins leads to genome modification rates of up to 26% in CD4(+) T cells and 17% in CD34(+) hematopoietic stem/progenitor cells. In addition, we show that multi-NLS ZFN proteins attenuate off-target effects and that codelivery of ZFN protein pairs facilitates dual gene modification frequencies of 20-30% in CD4(+) T cells. These results illustrate the applicability of ZFN protein delivery for precision genome engineering.
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http://dx.doi.org/10.1038/mtna.2015.6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354341PMC
March 2015

Genome engineering with custom recombinases.

Methods Enzymol 2014 ;546:79-91

Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA; Department of Cell and Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.

Site-specific recombinases are valuable tools for myriad basic research and genome engineering applications. In particular, hybrid recombinases consisting of catalytic domains from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are capable of introducing targeted modifications into mammalian cells. Due to their inherent modularity, new recombinases with distinct targeting specificities can readily be generated and utilized in a "plug-and-play" manner. In this protocol, we provide detailed, step-by-step instructions for generating new hybrid recombinases with user-defined specificity, as well as methods for achieving site-specific integration into targeted genomic loci using these systems.
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http://dx.doi.org/10.1016/B978-0-12-801185-0.00004-0DOI Listing
July 2015

Site-selective labeling of a lysine residue in human serum albumin.

Angew Chem Int Ed Engl 2014 Oct 4;53(44):11783-6. Epub 2014 Sep 4.

The Skaggs Institute for Chemical Biology, Department of Chemistry, and Department of Molecular and Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA).

Conjugation to human serum albumin (HSA) has emerged as a powerful approach for extending the in vivo half-life of many small molecule and peptide/protein drugs. Current HSA conjugation strategies, however, can often yield heterogeneous mixtures with inadequate pharmacokinetics, low efficacies, and variable safety profiles. Here, we designed and synthesized analogues of TAK-242, a small molecule inhibitor of Toll-like receptor 4, that primarily reacted with a single lysine residue of HSA. These TAK-242-based cyclohexene compounds demonstrated robust reactivity, and Lys64 was identified as the primary conjugation site. A bivalent HSA conjugate was also prepared in a site-specific manner. Additionally, HSA-cyclohexene conjugates maintained higher levels of stability both in human plasma and in mice than the corresponding maleimide conjugates. This new conjugation strategy promises to broadly enhance the performance of HSA conjugates for numerous applications.
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http://dx.doi.org/10.1002/anie.201405924DOI Listing
October 2014

Protein delivery using Cys2-His2 zinc-finger domains.

ACS Chem Biol 2014 Aug 19;9(8):1662-7. Epub 2014 Jun 19.

The Skaggs Institute for Chemical Biology and the Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute , La Jolla, California 92037, United States.

The development of new methods for delivering proteins into cells is a central challenge for advancing both basic research and therapeutic applications. We previously reported that zinc-finger nuclease proteins are intrinsically cell-permeable due to the cell-penetrating activity of the Cys2-His2 zinc-finger domain. Here, we demonstrate that genetically fused zinc-finger motifs can transport proteins and enzymes into a wide range of primary and transformed mammalian cell types. We show that zinc-finger domains mediate protein uptake at efficiencies that exceed conventional protein transduction systems and do so without compromising enzyme activity. In addition, we demonstrate that zinc-finger proteins enter cells primarily through macropinocytosis and facilitate high levels of cytosolic delivery. These findings establish zinc-finger proteins as not only useful tools for targeted genome engineering but also effective reagents for protein delivery.
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http://dx.doi.org/10.1021/cb500282gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519095PMC
August 2014

Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies.

Acc Chem Res 2014 Aug 30;47(8):2309-18. Epub 2014 May 30.

Department of Biomedical Engineering, Duke University , Durham, North Carolina 27708, United States.

The understanding of gene regulation and the structure and function of the human genome increased dramatically at the end of the 20th century. Yet the technologies for manipulating the genome have been slower to develop. For instance, the field of gene therapy has been focused on correcting genetic diseases and augmenting tissue repair for more than 40 years. However, with the exception of a few very low efficiency approaches, conventional genetic engineering methods have only been able to add auxiliary genes to cells. This has been a substantial obstacle to the clinical success of gene therapies and has also led to severe unintended consequences in several cases. Therefore, technologies that facilitate the precise modification of cellular genomes have diverse and significant implications in many facets of research and are essential for translating the products of the Genomic Revolution into tangible benefits for medicine and biotechnology. To address this need, in the 1990s, we embarked on a mission to develop technologies for engineering protein-DNA interactions with the aim of creating custom tools capable of targeting any DNA sequence. Our goal has been to allow researchers to reach into genomes to specifically regulate, knock out, or replace any gene. To realize these goals, we initially focused on understanding and manipulating zinc finger proteins. In particular, we sought to create a simple and straightforward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins using only defined modular components, a web-based utility, and standard recombinant DNA technology. Two significant challenges we faced were (i) the development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and (ii) determining how individual zinc finger domains could be tethered together as polydactyl proteins to recognize unique locations within complex genomes. We and others have since used this modular assembly method to engineer artificial proteins and enzymes that activate, repress, or create defined changes to user-specified genes in human cells, plants, and other organisms. We have also engineered novel methods for externally controlling protein activity and delivery, as well as developed new strategies for the directed evolution of protein and enzyme function. This Account summarizes our work in these areas and highlights independent studies that have successfully used the modular assembly approach to create proteins with novel function. We also discuss emerging alternative methods for genomic targeting, including transcription activator-like effectors (TALEs) and CRISPR/Cas systems, and how they complement the synthetic zinc finger protein technology.
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http://dx.doi.org/10.1021/ar500039wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139171PMC
August 2014

Enhancing the specificity of recombinase-mediated genome engineering through dimer interface redesign.

J Am Chem Soc 2014 Apr 20;136(13):5047-56. Epub 2014 Mar 20.

The Skaggs Institute for Chemical Biology and the Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute , La Jolla, California 92037, United States.

Despite recent advances in genome engineering made possible by the emergence of site-specific endonucleases, there remains a need for tools capable of specifically delivering genetic payloads into the human genome. Hybrid recombinases based on activated catalytic domains derived from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are a class of reagents capable of achieving this. The utility of these enzymes, however, has been constrained by their low overall targeting specificity, largely due to the formation of side-product homodimers capable of inducing off-target modifications. Here, we combine rational design and directed evolution to re-engineer the serine recombinase dimerization interface and generate a recombinase architecture that reduces formation of these undesirable homodimers by >500-fold. We show that these enhanced recombinases demonstrate substantially improved targeting specificity in mammalian cells and achieve rates of site-specific integration similar to those previously reported for site-specific nucleases. Additionally, we show that enhanced recombinases exhibit low toxicity and promote the delivery of the human coagulation factor IX and α-galactosidase genes into endogenous genomic loci with high specificity. These results provide a general means for improving hybrid recombinase specificity by protein engineering and illustrate the potential of these enzymes for basic research and therapeutic applications.
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http://dx.doi.org/10.1021/ja4130059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985937PMC
April 2014

Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering.

PLoS One 2014 20;9(1):e85755. Epub 2014 Jan 20.

The Departments of Chemistry and Cell and Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America.

Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human CCR5 and BMPR1A genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085755PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896395PMC
January 2015

Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants.

Nucleic Acids Res 2014 Apr 21;42(7):4755-66. Epub 2014 Jan 21.

The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA, Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA and Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

The serine recombinases are a diverse family of modular enzymes that promote high-fidelity DNA rearrangements between specific target sites. Replacement of their native DNA-binding domains with custom-designed Cys₂-His₂ zinc-finger proteins results in the creation of engineered zinc-finger recombinases (ZFRs) capable of achieving targeted genetic modifications. The flexibility afforded by zinc-finger domains enables the design of hybrid recombinases that recognize a wide variety of potential target sites; however, this technology remains constrained by the strict recognition specificities imposed by the ZFR catalytic domains. In particular, the ability to fully reprogram serine recombinase catalytic specificity has been impeded by conserved base requirements within each recombinase target site and an incomplete understanding of the factors governing DNA recognition. Here we describe an approach to complement the targeting capacity of ZFRs. Using directed evolution, we isolated mutants of the β and Sin recombinases that specifically recognize target sites previously outside the scope of ZFRs. Additionally, we developed a genetic screen to determine the specific base requirements for site-specific recombination and showed that specificity profiling enables the discovery of unique genomic ZFR substrates. Finally, we conducted an extensive and family-wide mutational analysis of the serine recombinase DNA-binding arm region and uncovered a diverse network of residues that confer target specificity. These results demonstrate that the ZFR repertoire is extensible and highlights the potential of ZFRs as a class of flexible tools for targeted genome engineering.
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http://dx.doi.org/10.1093/nar/gkt1389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985619PMC
April 2014

Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

ACS Synth Biol 2014 Oct 22;3(10):723-30. Epub 2013 Nov 22.

The Skaggs Institute for Chemical Biology and the Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute , La Jolla, California 92037, United States.

The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.
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http://dx.doi.org/10.1021/sb400114pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097969PMC
October 2014
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