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1.
Nat Med ; 25(2): 229-233, 2019 02.
Article in English | MEDLINE | ID: mdl-30664785

ABSTRACT

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.


Subject(s)
Gene Editing , Leber Congenital Amaurosis/genetics , Leber Congenital Amaurosis/physiopathology , Animals , Cell Line , Gene Knock-In Techniques , Humans , Mice , Primates , Reproducibility of Results , Vision, Ocular
2.
Nat Commun ; 9(1): 3542, 2018 08 28.
Article in English | MEDLINE | ID: mdl-30154463

ABSTRACT

The original HTML version of this Article incorrectly listed an affiliation of Josh Tycko as 'Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA', instead of the correct 'Present address: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA'. It also incorrectly listed an affiliation of this author as 'Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA, 02451, USA'.The original HTML version incorrectly listed an affiliation of Luis A. Barrera as 'Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06511, USA', instead of the correct 'Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA 02451, USA'.Finally, the original HTML version incorrectly omitted an affiliation of Nicholas C. Huston: 'Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA'.This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication.

3.
Nat Commun ; 9(1): 2962, 2018 07 27.
Article in English | MEDLINE | ID: mdl-30054474

ABSTRACT

Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences are generally more active, although high efficiency 20-mer spacers are markedly less tolerant of mismatches. Using this dataset, we developed an SaCas9 specificity model that performs robustly in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.


Subject(s)
CRISPR-Associated Protein 9/genetics , Gene Editing/methods , Gene Library , Staphylococcus aureus/genetics , Bacterial Proteins/genetics , Base Sequence , CRISPR-Cas Systems , Cloning, Molecular , Clustered Regularly Interspaced Short Palindromic Repeats , Genes, Bacterial/genetics , HEK293 Cells , Humans , RNA, Guide, Kinetoplastida/genetics
4.
PLoS Genet ; 12(4): e1005956, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27070429

ABSTRACT

To understand the molecular processes underlying aging, we screened modENCODE ChIP-seq data to identify transcription factors that bind to age-regulated genes in C. elegans. The most significant hit was the GATA transcription factor encoded by elt-2, which is responsible for inducing expression of intestinal genes during embryogenesis. Expression of ELT-2 decreases during aging, beginning in middle age. We identified genes regulated by ELT-2 in the intestine during embryogenesis, and then showed that these developmental genes markedly decrease in expression as worms grow old. Overexpression of elt-2 extends lifespan and slows the rate of gene expression changes that occur during normal aging. Thus, our results identify the developmental regulator ELT-2 as a major driver of normal aging in C. elegans.


Subject(s)
Aging/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/physiology , GATA Transcription Factors/genetics , Animals , Caenorhabditis elegans/genetics , Gene Expression Regulation/genetics , Intestines/growth & development , Mutation , Transcription, Genetic
5.
Genome Biol ; 16: 257, 2015 Nov 24.
Article in English | MEDLINE | ID: mdl-26596280

ABSTRACT

BACKGROUND: CRISPR-Cas systems have been broadly embraced as effective tools for genome engineering applications, with most studies to date utilizing the Streptococcus pyogenes Cas9. Here we characterize and manipulate the smaller, 1053 amino acid nuclease Staphylococcus aureus Cas9. RESULTS: We find that the S. aureus Cas9 recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at high efficiency with a variety of guide RNA (gRNA) spacer lengths. When directed against genomic targets with mutually permissive NGGRRT PAMs, the S. pyogenes Cas9 and S. aureus Cas9 yield indels at comparable rates. We additionally show D10A and N580A paired nickase activity with S. aureus Cas9, and we further package it with two gRNAs in a single functional adeno-associated virus (AAV) vector. Finally, we assess comparative S. pyogenes and S. aureus Cas9 specificity using GUIDE-seq. CONCLUSION: Our results reveal an S. aureus Cas9 that is effective for a variety of genome engineering purposes, including paired nickase approaches and all-in-one delivery of Cas9 and multiple gRNA expression cassettes with AAV vectors.


Subject(s)
Bacterial Proteins/genetics , CRISPR-Cas Systems/genetics , Endodeoxyribonucleases/genetics , Gene Transfer Techniques , Genetic Engineering , Staphylococcus aureus/genetics , Bacterial Proteins/metabolism , CRISPR-Associated Protein 9 , Deoxyribonuclease I/genetics , Dependovirus/genetics , Endonucleases/genetics , Endonucleases/metabolism , RNA Editing , Streptococcus pyogenes/genetics
6.
Methods Mol Biol ; 1327: 59-74, 2015.
Article in English | MEDLINE | ID: mdl-26423968

ABSTRACT

The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe a method using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome.


Subject(s)
CRISPR-Cas Systems/genetics , Caenorhabditis elegans/genetics , Genetic Engineering/methods , Genome , Animals , Animals, Genetically Modified , Cloning, Molecular , Gene Targeting/methods , INDEL Mutation , Plasmids/genetics , RNA, Guide, Kinetoplastida/genetics
7.
Genetics ; 195(3): 1181-5, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23979579

ABSTRACT

We adapted the CRISPR-Cas9 system for template-mediated repair of targeted double-strand breaks via homologous recombination in Caenorhabditis elegans, enabling customized and efficient genome editing. This system can be used to create specific insertions, deletions, and base pair changes in the germline of C. elegans.


Subject(s)
CRISPR-Cas Systems , Caenorhabditis elegans/genetics , Genome, Helminth , Animals , DNA Breaks, Double-Stranded , DNA, Helminth/genetics , Gene Targeting , Genetic Engineering , Homologous Recombination , Mutagenesis , Mutagenesis, Insertional , RNA Editing/genetics
8.
Nat Methods ; 10(8): 741-3, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23817069

ABSTRACT

We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.


Subject(s)
Caenorhabditis elegans/genetics , Genome , Inverted Repeat Sequences , RNA, Small Nuclear/genetics , Animals , Genetic Engineering/methods , Promoter Regions, Genetic , Zebrafish Proteins/genetics , RNA, Small Untranslated
9.
Science ; 324(5931): 1199-202, 2009 May 29.
Article in English | MEDLINE | ID: mdl-19478183

ABSTRACT

Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as counting. A cellular counter would enable complex synthetic programming and a variety of biotechnology applications. Here, we report two complementary synthetic genetic counters in Escherichia coli that can count up to three induction events: the first, a riboregulated transcriptional cascade, and the second, a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher numbers.


Subject(s)
DNA, Bacterial/genetics , Escherichia coli K12/genetics , Gene Regulatory Networks , Protein Biosynthesis , Recombinases/metabolism , Regulatory Elements, Transcriptional , Transcription, Genetic , Arabinose/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation, Bacterial , Genetic Engineering , Green Fluorescent Proteins/biosynthesis , Models, Genetic , Plasmids , Promoter Regions, Genetic , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Recombinases/genetics , Viral Proteins/genetics , Viral Proteins/metabolism
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