- Email: [email protected]
Cas Nucleases
Gene Editing and Gene Regulation I 58. Engineered Cas9 Variants with Novel PAM Specificities Expand the Targeting Range of CRISPR/Cas Nucleases
Benjamin P. Kleinstiver,1,2,3 Michelle S. Prew,1,2 Ved V. Topkar,1,2 Shengdar Q. Tsai,1,2,3 Jae K. Joung.1,2,3 1 Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA; 2Center for Cancer Research, Massachusetts General Hostpial, Charlestown, MA; 3Department of Pathology, Harvard Medical School, Boston, MA. Specificity and targeting range are important considerations when selecting an engineered nuclease platform for functional studies or therapeutic applications. The use of CRISPR/Cas9 to edit virtually any genome has enabled researchers to rapidly knock out genes or to introduce precise genomic changes. A number of studies have explored and improved the specificity of the CRISPR/Cas9 platform but the targeting range remains limited to certain sequences. The most widely used Cas9 from Streptococcus pyogenes (SpCas9) recognizes a protospacer adjacent motif (PAM) of the form NGG, a constraint that restricts the range of sequences that can be edited. Combined with the 5’ nucleotide requirement for guide-RNA expression from a polIII promoter, this restriction can be limiting particularly when one is using CRISPR/Cas9 nucleases to introduce precise mutations by homology-directed repair (HDR), which requires that double-strand breaks be introduced very closely to the site of sequence alteration. To address this challenge, we developed a heterologous genetic system that enabled us to rapidly screen libraries of Cas9 clones and identify variants with distinct cleavage specificities. Using this technology, we identified and optimized two different Cas9 variants that recognize novel PAMs. We demonstrate that these variants enable targeting of sites with non-canonical PAM sequences that were previously inaccessible with wild-type SpCas9, more than doubling the total range of sequences that can be edited by HDR and by errorprone non-homologous end-joining (NHEJ). We demonstrate that these variants can robustly modify endogenous genes in human cells and that they are compatible with previously described specificity improvements to SpCas9 such as truncated gRNAs. Furthermore, we also show Cas9 nucleases from other bacterial species such as Staphylococcus aureus and Streptococcus thermophilus can also be assessed for activity in our heterologous genetic system, suggesting that these alternative Cas9s might also be modified in their PAM specificities using the same approach we undertook with SpCas9. These orthogonal Cas9 nucleases offer the advantage of being substantially smaller in size than SpCas9, an important factor when considering packaging limits for viral delivery of CRISPR/Cas9 components. Taken together, our results more than double the targeting range of CRISPR/Cas9 nuclease platform and improve the prospects for translation of these reagents to clinical settings.
59. Multiplex Gene Activation by CRISPR/ Cas9-Based Transcription Factors for the Direct Conversion of Fibroblasts to a Neuronal Phenotype
Joshua Black,1 Andrew Adler,3 Hunter Hutchinson,1 Honggang Wang,1 Geoffrey Pitt,1 Kam Leong,2 Charles Gersbach.1 1 Duke University, Durham; 2Columbia University, New York; 3 University of California, San Diego, La Jolla. The reprogramming of cell lineage between mature somatic cell types has a significant potential to advance disease modeling, drug discovery, and gene and cell therapies for regenerative medicine. Several groups have established methods to reprogram cell phenotype through the ectopic delivery of cDNAs encoding master regulatory
S26
transcription factors. These factors can target epigenetically silenced genes and activate transcriptional networks specific to other cell lineages. The recent advances made in engineering the CRISPR/Cas9 system as a programmable transcriptional regulator provide an opportunity to study alternate methods to achieve cell reprogramming. The dCas9based transcription factors (dCas9-TFs) can attain multiplex activation and repression of endogenous genes. It’s simplicity of use enables a standardized method of regulating endogenous transcriptional networks in any cell type of interest. The ability of dCas9-TFs to target virtually any region of the genome within the native chromatin context provides an opportunity to study how non-coding regulatory elements and epigenetic signatures influence reprogramming outcomes. Here we demonstrate the capacity of dCas9-TFs to drive the direct conversion of murine embryonic fibroblasts (MEFs) to cells of a neuronal phenotype. We utilized a dCas9-TF with both N-terminal and C-terminal VP64 transactivation domains and demonstrated a 10-fold improvement in activation of target genes compared to dCas9-TFs with only a single VP64. Using this dCas9-TF variant, we demonstrated the multiplex activation of BRN2, ASCL1, and MYT1L (BAM factors) in MEFs. These three factors have been shown to generate functional neurons when expressed ectopically in MEFs. When compared to overexpression of the transgenes, dCas9-TFs induced significantly less mRNA and protein expression of the BAM factors, as determined by qRT-PCR and immunofluorescence staining. However, by day 14 in culture following the delivery of dCas9-TFs targeting the endogenous BAM factors, we identified cells positive for the pan-neuronal markers TUJ1 and MAP2 that exhibited complex neuronal morphologies. Furthermore, we found that dCas9TFs were able to generate 23% more TUJ1/MAP2-co-positive cells than that achieved through the ectopic delivery of the BAM factors. Electrophysiological recordings of these cells identified single, action potential-like responses to step-depolarization by current clamp in the whole cell configuration. The cells also displayed calcium transients in response to KCl-induced depolarization, measured by monitoring fluorescence intensity of a GCaMP reporter. Ongoing work is focused on improving the functional maturity of the cells and addressing the kinetics of gene activation and epigenetic reprogramming at the target loci. We hypothesize that the targeted activation at the endogenous loci may more deterministically reprogram the chromatin to marks of active transcription that stabilize gene expression.
60. Intramuscular and Systemic Induction of the N-Truncated Dystrophin By Out-Of-Frame Exon 2 Skipping Restores Muscle Function in the Dup2 Mouse, Providing Further Support for a Therapeutic Pathway for 5’ DMD Mutations
Nicolas Wein,1 Adeline Vulin,1 Tabatha Simmons,1 Felecia Gumienny,1 Nianyuan Huang,1 Francesco Muntoni,2 James Ervasti,3 Robert Weiss,4 Kevin Flanigan.1 1 Center for Gene Therapy, Nationwidechildrens Hospital, Columbus, OH; 2Developmental Neuroscience, UCL Institute of Child Health, London, United Kingdom; 3Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN; 4Department of Human Genetics, The University of Utah School of Medicine, Salt Lake City, UT.
Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon. Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
Benjamin P. Kleinstiver,1,2,3 Michelle S. Prew,1,2 Ved V. Topkar,1,2 Shengdar Q. Tsai,1,2,3 Jae K. Joung.1,2,3 1 Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA; 2Center for Cancer Research, Massachusetts General Hostpial, Charlestown, MA; 3Department of Pathology, Harvard Medical School, Boston, MA. Specificity and targeting range are important considerations when selecting an engineered nuclease platform for functional studies or therapeutic applications. The use of CRISPR/Cas9 to edit virtually any genome has enabled researchers to rapidly knock out genes or to introduce precise genomic changes. A number of studies have explored and improved the specificity of the CRISPR/Cas9 platform but the targeting range remains limited to certain sequences. The most widely used Cas9 from Streptococcus pyogenes (SpCas9) recognizes a protospacer adjacent motif (PAM) of the form NGG, a constraint that restricts the range of sequences that can be edited. Combined with the 5’ nucleotide requirement for guide-RNA expression from a polIII promoter, this restriction can be limiting particularly when one is using CRISPR/Cas9 nucleases to introduce precise mutations by homology-directed repair (HDR), which requires that double-strand breaks be introduced very closely to the site of sequence alteration. To address this challenge, we developed a heterologous genetic system that enabled us to rapidly screen libraries of Cas9 clones and identify variants with distinct cleavage specificities. Using this technology, we identified and optimized two different Cas9 variants that recognize novel PAMs. We demonstrate that these variants enable targeting of sites with non-canonical PAM sequences that were previously inaccessible with wild-type SpCas9, more than doubling the total range of sequences that can be edited by HDR and by errorprone non-homologous end-joining (NHEJ). We demonstrate that these variants can robustly modify endogenous genes in human cells and that they are compatible with previously described specificity improvements to SpCas9 such as truncated gRNAs. Furthermore, we also show Cas9 nucleases from other bacterial species such as Staphylococcus aureus and Streptococcus thermophilus can also be assessed for activity in our heterologous genetic system, suggesting that these alternative Cas9s might also be modified in their PAM specificities using the same approach we undertook with SpCas9. These orthogonal Cas9 nucleases offer the advantage of being substantially smaller in size than SpCas9, an important factor when considering packaging limits for viral delivery of CRISPR/Cas9 components. Taken together, our results more than double the targeting range of CRISPR/Cas9 nuclease platform and improve the prospects for translation of these reagents to clinical settings.
59. Multiplex Gene Activation by CRISPR/ Cas9-Based Transcription Factors for the Direct Conversion of Fibroblasts to a Neuronal Phenotype
Joshua Black,1 Andrew Adler,3 Hunter Hutchinson,1 Honggang Wang,1 Geoffrey Pitt,1 Kam Leong,2 Charles Gersbach.1 1 Duke University, Durham; 2Columbia University, New York; 3 University of California, San Diego, La Jolla. The reprogramming of cell lineage between mature somatic cell types has a significant potential to advance disease modeling, drug discovery, and gene and cell therapies for regenerative medicine. Several groups have established methods to reprogram cell phenotype through the ectopic delivery of cDNAs encoding master regulatory
S26
transcription factors. These factors can target epigenetically silenced genes and activate transcriptional networks specific to other cell lineages. The recent advances made in engineering the CRISPR/Cas9 system as a programmable transcriptional regulator provide an opportunity to study alternate methods to achieve cell reprogramming. The dCas9based transcription factors (dCas9-TFs) can attain multiplex activation and repression of endogenous genes. It’s simplicity of use enables a standardized method of regulating endogenous transcriptional networks in any cell type of interest. The ability of dCas9-TFs to target virtually any region of the genome within the native chromatin context provides an opportunity to study how non-coding regulatory elements and epigenetic signatures influence reprogramming outcomes. Here we demonstrate the capacity of dCas9-TFs to drive the direct conversion of murine embryonic fibroblasts (MEFs) to cells of a neuronal phenotype. We utilized a dCas9-TF with both N-terminal and C-terminal VP64 transactivation domains and demonstrated a 10-fold improvement in activation of target genes compared to dCas9-TFs with only a single VP64. Using this dCas9-TF variant, we demonstrated the multiplex activation of BRN2, ASCL1, and MYT1L (BAM factors) in MEFs. These three factors have been shown to generate functional neurons when expressed ectopically in MEFs. When compared to overexpression of the transgenes, dCas9-TFs induced significantly less mRNA and protein expression of the BAM factors, as determined by qRT-PCR and immunofluorescence staining. However, by day 14 in culture following the delivery of dCas9-TFs targeting the endogenous BAM factors, we identified cells positive for the pan-neuronal markers TUJ1 and MAP2 that exhibited complex neuronal morphologies. Furthermore, we found that dCas9TFs were able to generate 23% more TUJ1/MAP2-co-positive cells than that achieved through the ectopic delivery of the BAM factors. Electrophysiological recordings of these cells identified single, action potential-like responses to step-depolarization by current clamp in the whole cell configuration. The cells also displayed calcium transients in response to KCl-induced depolarization, measured by monitoring fluorescence intensity of a GCaMP reporter. Ongoing work is focused on improving the functional maturity of the cells and addressing the kinetics of gene activation and epigenetic reprogramming at the target loci. We hypothesize that the targeted activation at the endogenous loci may more deterministically reprogram the chromatin to marks of active transcription that stabilize gene expression.
60. Intramuscular and Systemic Induction of the N-Truncated Dystrophin By Out-Of-Frame Exon 2 Skipping Restores Muscle Function in the Dup2 Mouse, Providing Further Support for a Therapeutic Pathway for 5’ DMD Mutations
Nicolas Wein,1 Adeline Vulin,1 Tabatha Simmons,1 Felecia Gumienny,1 Nianyuan Huang,1 Francesco Muntoni,2 James Ervasti,3 Robert Weiss,4 Kevin Flanigan.1 1 Center for Gene Therapy, Nationwidechildrens Hospital, Columbus, OH; 2Developmental Neuroscience, UCL Institute of Child Health, London, United Kingdom; 3Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN; 4Department of Human Genetics, The University of Utah School of Medicine, Salt Lake City, UT.
Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon. Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy