- Email: [email protected]
489. Quantifying Rates of Gene Targeting and Off-Target Cleavage of Engineered Nucleases Using SMRT Sequencing
NUCLEASE MEDIATED GENOME EDITING principle establishing phenotypic correction of hemophilia B, and show that appropriately designed donors may expand this strategy to treat a range of monogenic diseases.
489. Quantifying Rates of Gene Targeting and Off-Target Cleavage of Engineered Nucleases Using SMRT Sequencing
Eli J. Fine,1 Eric Kildebeck,2 Yanni Lin,1 Matthew H. Porteus,2 Thomas J. Cradick,1 Gang Bao.1 1 Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, GA; 2Pediatrics, Stanford University, Palo Alto, CA. Transcription Activator-Like Effector Nucleases (TALENs) are a powerful tool for genome engineering since they can be designed to target a DNA sequence in a modular fashion for specific DNA cleavage in a given genome. Repair of this lesion through the nonhomologous end-joining (NHEJ) pathway can result in a targeted knockout of a gene of interest. Alternatively, homology directed repair (HDR) using an exogenously supplied DNA template can result in targeted gene addition or modification. TALENs have been used to create model organisms, custom cell lines, and are in development for gene therapy. However, discriminating between similar targets in the same gene family has been a challenge for engineered nucleases; the CCR5 zinc finger nucleases in clinical trials have only a 4:1 preference for cleaving CCR5 over CCR2. Quantifying rates of gene targeting in primary cells is another major challenge due to the low frequency of these events. To date, the commonly used techniques for assessing the efficacy of TALENs are the Surveyor Nuclease Assay and Restriction Fragment Length Polymorphism (RFLP). Both techniques are well suited for detecting modifications at rates as low as a few percent. However, for detecting low-frequency off-target events or low, but clinically relevant, rates of HDR in primary cells, these techniques are not effective. Deep sequencing can be used to detect low-frequency off-target events and the analysis of single cell clones can detect low rates of HDR, but both of these methods are difficult and expensive. To address these challenges, we employed Single Molecule Real-Time (SMRT) sequencing: a 3rd generation platform that offers long read lengths, high fidelity, reasonable sensitivity, and variable sequencing depth at a relatively low cost. We created TALENs targeting the beta-globin gene near the sickle-cell mutation with cleavage rates of 62% in 293T cells. Using SMRT sequencing, we found that these TALENs cleave a highly similar sequence in the delta-globin gene at a rate less than 0.58% (p < 0.05), demonstrating a greater than 100 fold preference for cleaving beta-globin over delta-globin. Using a different set of TALENs, we were also able to detect rates of targeting cDNA to the IL2Rg locus in primary human CD34+ cells of 1%. Cleavage at off-target sites by engineered nucleases is a large concern. Previously, only three TALEN off-target sites had been identified, all through using SELEX as a predictive assay and deep sequencing to confirm cleavage at the off-target site. We used the newly developed PROGNOS algorithm to predict potential off-target sites for TALENs with different repeat variable di-residues to target guanine (NK, NN, and NH) and analyzed the cleavage sites from transfected cells using SMRT sequencing. We identified several off-target sites for these novel nucleases, some with homology to the intended target as low as 76% and others lacking a 5’ pyrimidine. The use of SMRT sequencing as an assay format for genomic modifications induced by engineered nucleases holds great promise for allowing detection of low frequency events.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
490. Precise Modification of Human Genes Directed by Single-Stranded DNA Oligonucleotides and TALENs
Shengdar Q. Tsai,1,2 Deepak Reyon,1,2 Cyd Khayter,1 J. Keith Joung.1,2 1 Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA; 2Department of Pathology, Harvard Medical School, Boston, MA.
The capability to direct precise modifications to the genome could broadly enhance biological research, and in the longer term, enable therapies of genetic diseases. Single-stranded DNA oligonucleotides (ssODNs) have been used with engineered nucleases to introduce precise mutations into endogenous genes. While ssODNs have been optimized with zinc finger nucleases (ZFNs), the robustness of ssODN-mediated repair with TAL effector nucleases (TALENs) has not been systematically optimized. We assessed the effect of ssODN homology length on the efficiency of gene alteration using an EGFP reporter repair assay and then used optimal-length ssODNs with TALENs to successfully direct precise nucleotide insertions or substitutions to sites in 13 human endogenous genes. The high frequency of sequence modifications we observed enabled us to isolate precisely modified single-cell clones without the need for selection. This high efficiency of modification coupled with the extended targeting range of TALENs compared to ZFNs make ssODN/TALENdirected repair an attractive approach for simple, rapid, and precise genome modification.
491. Efficient Template-Free Genetic Correction with TALE Nucleases
David G. Ousterout,1 Pablo Perez-Pinera,1 Pratiksha I. Thakore,1 Ami M. Kabadi,1 Matthew T. Brown,1 Kamel Mamchaoui,2 Jacques P. Tremblay,3 Charles A. Gersbach.1,4 1 Biomedical Engineering, Duke University, Durham, NC; 2Institut de Myologie, Paris, France; 3Unité de Recherche de Recherche en Génétique Humaine, Université Laval, Quebec, Canada; 4Institute for Genome Sciences and Policy, Duke University, Durham, NC. Genome editing with engineered nucleases is a powerful approach for correcting genetic mutations. Current strategies exclusively utilize homologous recombination guided by a synthetic DNA repair template. Although this approach is extremely valuable for restoring the complete coding sequence of the affected gene, simply restoring a disrupted reading frame or removing a premature stop codon could ameliorate the symptoms of some genetic diseases. Therefore, frame restoration could be accomplished by utilizing the stochastic, error-prone non-homologous end-joining DNA repair pathway that creates small insertions and deletions of random length at the targeted DNA break point. Here we demonstrate the restoration of dystrophin expression in cells from Duchenne muscular dystrophy (DMD) patients with transcription activator-like effector nucleases (TALENs) in the absence of a DNA template by utilizing homologyindependent repair pathways to correct disrupted reading frames. TALENs were targeted to exon 51 of the dystrophin gene with the potential to correct aberrant reading frames occurring in up to 8.4% of known DMD mutations. First, several combinations of TALEN pairs targeted to exon 51 were screened for optimal activity and minimal cytotoxicity in human cells. Using the optimal TALEN pair, we demonstrated that reading frame restoration after TALEN treatment rescued dystrophin expression in corrected clonal populations of skeletal myoblasts from DMD patients. Furthermore, this TALEN mediated high efficiency gene editing of up to 12.7% of alleles and restored detectable levels of dystrophin protein expression in a bulktreated population of DMD muscle cells without any selection or clonal isolation. This TALEN also modified up to 5.5% of alleles in S189
489. Quantifying Rates of Gene Targeting and Off-Target Cleavage of Engineered Nucleases Using SMRT Sequencing
Eli J. Fine,1 Eric Kildebeck,2 Yanni Lin,1 Matthew H. Porteus,2 Thomas J. Cradick,1 Gang Bao.1 1 Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, GA; 2Pediatrics, Stanford University, Palo Alto, CA. Transcription Activator-Like Effector Nucleases (TALENs) are a powerful tool for genome engineering since they can be designed to target a DNA sequence in a modular fashion for specific DNA cleavage in a given genome. Repair of this lesion through the nonhomologous end-joining (NHEJ) pathway can result in a targeted knockout of a gene of interest. Alternatively, homology directed repair (HDR) using an exogenously supplied DNA template can result in targeted gene addition or modification. TALENs have been used to create model organisms, custom cell lines, and are in development for gene therapy. However, discriminating between similar targets in the same gene family has been a challenge for engineered nucleases; the CCR5 zinc finger nucleases in clinical trials have only a 4:1 preference for cleaving CCR5 over CCR2. Quantifying rates of gene targeting in primary cells is another major challenge due to the low frequency of these events. To date, the commonly used techniques for assessing the efficacy of TALENs are the Surveyor Nuclease Assay and Restriction Fragment Length Polymorphism (RFLP). Both techniques are well suited for detecting modifications at rates as low as a few percent. However, for detecting low-frequency off-target events or low, but clinically relevant, rates of HDR in primary cells, these techniques are not effective. Deep sequencing can be used to detect low-frequency off-target events and the analysis of single cell clones can detect low rates of HDR, but both of these methods are difficult and expensive. To address these challenges, we employed Single Molecule Real-Time (SMRT) sequencing: a 3rd generation platform that offers long read lengths, high fidelity, reasonable sensitivity, and variable sequencing depth at a relatively low cost. We created TALENs targeting the beta-globin gene near the sickle-cell mutation with cleavage rates of 62% in 293T cells. Using SMRT sequencing, we found that these TALENs cleave a highly similar sequence in the delta-globin gene at a rate less than 0.58% (p < 0.05), demonstrating a greater than 100 fold preference for cleaving beta-globin over delta-globin. Using a different set of TALENs, we were also able to detect rates of targeting cDNA to the IL2Rg locus in primary human CD34+ cells of 1%. Cleavage at off-target sites by engineered nucleases is a large concern. Previously, only three TALEN off-target sites had been identified, all through using SELEX as a predictive assay and deep sequencing to confirm cleavage at the off-target site. We used the newly developed PROGNOS algorithm to predict potential off-target sites for TALENs with different repeat variable di-residues to target guanine (NK, NN, and NH) and analyzed the cleavage sites from transfected cells using SMRT sequencing. We identified several off-target sites for these novel nucleases, some with homology to the intended target as low as 76% and others lacking a 5’ pyrimidine. The use of SMRT sequencing as an assay format for genomic modifications induced by engineered nucleases holds great promise for allowing detection of low frequency events.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
490. Precise Modification of Human Genes Directed by Single-Stranded DNA Oligonucleotides and TALENs
Shengdar Q. Tsai,1,2 Deepak Reyon,1,2 Cyd Khayter,1 J. Keith Joung.1,2 1 Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA; 2Department of Pathology, Harvard Medical School, Boston, MA.
The capability to direct precise modifications to the genome could broadly enhance biological research, and in the longer term, enable therapies of genetic diseases. Single-stranded DNA oligonucleotides (ssODNs) have been used with engineered nucleases to introduce precise mutations into endogenous genes. While ssODNs have been optimized with zinc finger nucleases (ZFNs), the robustness of ssODN-mediated repair with TAL effector nucleases (TALENs) has not been systematically optimized. We assessed the effect of ssODN homology length on the efficiency of gene alteration using an EGFP reporter repair assay and then used optimal-length ssODNs with TALENs to successfully direct precise nucleotide insertions or substitutions to sites in 13 human endogenous genes. The high frequency of sequence modifications we observed enabled us to isolate precisely modified single-cell clones without the need for selection. This high efficiency of modification coupled with the extended targeting range of TALENs compared to ZFNs make ssODN/TALENdirected repair an attractive approach for simple, rapid, and precise genome modification.
491. Efficient Template-Free Genetic Correction with TALE Nucleases
David G. Ousterout,1 Pablo Perez-Pinera,1 Pratiksha I. Thakore,1 Ami M. Kabadi,1 Matthew T. Brown,1 Kamel Mamchaoui,2 Jacques P. Tremblay,3 Charles A. Gersbach.1,4 1 Biomedical Engineering, Duke University, Durham, NC; 2Institut de Myologie, Paris, France; 3Unité de Recherche de Recherche en Génétique Humaine, Université Laval, Quebec, Canada; 4Institute for Genome Sciences and Policy, Duke University, Durham, NC. Genome editing with engineered nucleases is a powerful approach for correcting genetic mutations. Current strategies exclusively utilize homologous recombination guided by a synthetic DNA repair template. Although this approach is extremely valuable for restoring the complete coding sequence of the affected gene, simply restoring a disrupted reading frame or removing a premature stop codon could ameliorate the symptoms of some genetic diseases. Therefore, frame restoration could be accomplished by utilizing the stochastic, error-prone non-homologous end-joining DNA repair pathway that creates small insertions and deletions of random length at the targeted DNA break point. Here we demonstrate the restoration of dystrophin expression in cells from Duchenne muscular dystrophy (DMD) patients with transcription activator-like effector nucleases (TALENs) in the absence of a DNA template by utilizing homologyindependent repair pathways to correct disrupted reading frames. TALENs were targeted to exon 51 of the dystrophin gene with the potential to correct aberrant reading frames occurring in up to 8.4% of known DMD mutations. First, several combinations of TALEN pairs targeted to exon 51 were screened for optimal activity and minimal cytotoxicity in human cells. Using the optimal TALEN pair, we demonstrated that reading frame restoration after TALEN treatment rescued dystrophin expression in corrected clonal populations of skeletal myoblasts from DMD patients. Furthermore, this TALEN mediated high efficiency gene editing of up to 12.7% of alleles and restored detectable levels of dystrophin protein expression in a bulktreated population of DMD muscle cells without any selection or clonal isolation. This TALEN also modified up to 5.5% of alleles in S189