Rationally Engineered Cas9 Nucleases With Improved Specificity
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In this paper, the authors used structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9) using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells.Abstract:
The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.read more
Citations
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Journal ArticleDOI
Genome engineering through CRISPR/Cas9 technology in the human germline and pluripotent stem cells
TL;DR: This narrative review seeks to understand the possible impact of CRISR/Cas9 technology on human reproduction from the technical and ethical point of view, and suggest a course of action for the scientific community.
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A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells.
Sara E. Howden,Sara E. Howden,Bradley McColl,Astrid Glaser,Astrid Glaser,Jim Vadolas,Jim Vadolas,Steven Petrou,Melissa H. Little,Melissa H. Little,Andrew G. Elefanty,Edouard G. Stanley,Edouard G. Stanley,Edouard G. Stanley +13 more
TL;DR: It is demonstrated that transient SpCas9-Gem expression enables reliable generation of both knockin reporter cell lines and genetically repaired patient-specific induced pluripotent stem cell lines free of unwanted mutations at the targeted locus.
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The CRISPR/Cas9 system and its applications in crop genome editing
TL;DR: An overview of the CRISPR/Cas9 system itself is presented, including its mechanism of action, system construction strategies, and the screening methods used to identify mutants containing edited genes.
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CRISPR/Cas-based genome engineering in natural product discovery
TL;DR: This review briefly introduces and summarizes current knowledge about the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR/Cas system and how it was engineered to become one of the most important and versatile genome editing techniques that is currently revolutionizing the whole field of molecular biology.
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Generating mouse models for biomedical research: technological advances
TL;DR: Newer molecular technologies to precisely and efficiently manipulate the mammalian genome are enabling the production of more scientifically valuable animal models.
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Multiplex Genome Engineering Using CRISPR/Cas Systems
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Le Cong,F. A. Ran,David Benjamin Turitz Cox,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +10 more
TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
Journal ArticleDOI
RNA-Guided Human Genome Engineering via Cas9
Prashant Mali,Luhan Yang,Kevin M. Esvelt,John Aach,Marc Güell,James E. DiCarlo,Julie E. Norville,George M. Church,George M. Church +8 more
TL;DR: The type II bacterial CRISPR system is engineer to function with custom guide RNA (gRNA) in human cells to establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
Journal ArticleDOI
DNA targeting specificity of RNA-guided Cas9 nucleases
Patrick D. Hsu,David A. Scott,David A. Scott,Joshua A. Weinstein,Joshua A. Weinstein,F. Ann Ran,F. Ann Ran,F. Ann Ran,Silvana Konermann,Silvana Konermann,Vineeta Agarwala,Vineeta Agarwala,Vineeta Agarwala,Yinqing Li,Yinqing Li,Eli J. Fine,Xuebing Wu,Ophir Shalem,Ophir Shalem,Thomas J. Cradick,Luciano A. Marraffini,Gang Bao,Feng Zhang,Feng Zhang +23 more
TL;DR: In this article, the Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing.
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