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  • 1
    Publication Date: 2015-06-23
    Description: Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540238/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540238/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Kleinstiver, Benjamin P -- Prew, Michelle S -- Tsai, Shengdar Q -- Topkar, Ved V -- Nguyen, Nhu T -- Zheng, Zongli -- Gonzales, Andrew P W -- Li, Zhuyun -- Peterson, Randall T -- Yeh, Jing-Ruey Joanna -- Aryee, Martin J -- Joung, J Keith -- DP1 GM105378/DP/NCCDPHP CDC HHS/ -- DP1 GM105378/GM/NIGMS NIH HHS/ -- R01 GM088040/GM/NIGMS NIH HHS/ -- R01 GM107427/GM/NIGMS NIH HHS/ -- England -- Nature. 2015 Jul 23;523(7561):481-5. doi: 10.1038/nature14592. Epub 2015 Jun 22.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉1] Molecular Pathology Unit &Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [3] Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA. ; 1] Molecular Pathology Unit &Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. ; 1] Molecular Pathology Unit &Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA [3] Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm SE-171 77, Sweden. ; 1] Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA [3] Broad Institute, Cambridge, Massachusetts 02142, USA. ; Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. ; 1] Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA. ; 1] Molecular Pathology Unit &Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA [3] Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26098369" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Substitution/genetics ; Animals ; CRISPR-Associated Proteins/*genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Directed Molecular Evolution ; Genome/genetics ; Humans ; Mutation/genetics ; *Nucleotide Motifs ; Protein Engineering/*methods ; Staphylococcus aureus/enzymology ; Streptococcus pyogenes/*enzymology ; Streptococcus thermophilus/enzymology ; Substrate Specificity/genetics ; Zebrafish/embryology/genetics
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 2
    Publication Date: 2014-01-11
    Description: It has been assumed that most, if not all, signals regulating early development have been identified. Contrary to this expectation, we identified 28 candidate signaling proteins expressed during zebrafish embryogenesis, including Toddler, a short, conserved, and secreted peptide. Both absence and overproduction of Toddler reduce the movement of mesendodermal cells during zebrafish gastrulation. Local and ubiquitous production of Toddler promote cell movement, suggesting that Toddler is neither an attractant nor a repellent but acts globally as a motogen. Toddler drives internalization of G protein-coupled APJ/Apelin receptors, and activation of APJ/Apelin signaling rescues toddler mutants. These results indicate that Toddler is an activator of APJ/Apelin receptor signaling, promotes gastrulation movements, and might be the first in a series of uncharacterized developmental signals.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107353/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107353/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Pauli, Andrea -- Norris, Megan L -- Valen, Eivind -- Chew, Guo-Liang -- Gagnon, James A -- Zimmerman, Steven -- Mitchell, Andrew -- Ma, Jiao -- Dubrulle, Julien -- Reyon, Deepak -- Tsai, Shengdar Q -- Joung, J Keith -- Saghatelian, Alan -- Schier, Alexander F -- K99 HD076935/HD/NICHD NIH HHS/ -- R01 GM056211/GM/NIGMS NIH HHS/ -- R01 GM102491/GM/NIGMS NIH HHS/ -- R01 HG005111/HG/NHGRI NIH HHS/ -- Howard Hughes Medical Institute/ -- New York, N.Y. -- Science. 2014 Feb 14;343(6172):1248636. doi: 10.1126/science.1248636. Epub 2014 Jan 9.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/24407481" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Sequence ; Animals ; *Cell Movement ; Chemokine CXCL12/metabolism ; Frameshift Mutation ; Gastrulation/genetics/*physiology ; Molecular Sequence Data ; Receptors, G-Protein-Coupled/genetics/*metabolism ; Signal Transduction ; Zebrafish/*embryology/genetics/metabolism ; Zebrafish Proteins/genetics/*metabolism
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 3
    Publication Date: 2016-01-07
    Description: CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with 〉85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Kleinstiver, Benjamin P -- Pattanayak, Vikram -- Prew, Michelle S -- Tsai, Shengdar Q -- Nguyen, Nhu T -- Zheng, Zongli -- Joung, J Keith -- DP1 GM105378/DP/NCCDPHP CDC HHS/ -- R01 GM088040/GM/NIGMS NIH HHS/ -- R01 GM107427/GM/NIGMS NIH HHS/ -- England -- Nature. 2016 Jan 28;529(7587):490-5. doi: 10.1038/nature16526. Epub 2016 Jan 6.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. ; Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA. ; Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26735016" target="_blank"〉PubMed〈/a〉
    Keywords: Base Sequence ; CRISPR-Associated Proteins/*genetics/*metabolism ; CRISPR-Cas Systems/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/genetics/metabolism ; DNA-Binding Proteins/genetics/metabolism ; Endonucleases/genetics/*metabolism ; *Genetic Engineering ; Genome, Human/*genetics ; Humans ; Mutation ; Protein Binding ; RNA/genetics ; Reproducibility of Results ; Sequence Analysis, DNA ; Streptococcus pyogenes/enzymology/genetics ; Substrate Specificity
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 4
    Publication Date: 2012-09-13
    Description: Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 5
    Publication Date: 2014-01-09
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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