Rationally Engineered Cas9 Nucleases With Improved Specificity
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TLDR
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
Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways
Aziz Taghbalout,Menghan Du,Nathaniel Jillette,Wojciech Rosikiewicz,Abhijit Rath,Christopher D. Heinen,Sheng Li,Albert W. Cheng,Albert W. Cheng +8 more
TL;DR: A toolbox for editing DNA methylation to enable research investigations interrogating DNA methylomes is established and a stable and expression-inducible system for broader application of the Casilio-ME platforms is expanded.
Journal ArticleDOI
Rapid Generation of Somatic Mouse Mosaics with Locus-Specific, Stably Integrated Transgenic Elements.
Gi Bum Kim,David Rincon Fernandez Pacheco,David Saxon,Amy Yang,Sara Sabet,Marina Dutra-Clarke,Rachelle Levy,Ashley Watkins,Hannah Park,Aslam Abbasi Akhtar,Paul W. Linesch,Naomi Kobritz,Swasty S. Chandra,Katie Grausam,Alberto E. Ayala-Sarmiento,Jessica Molina,Kristyna Sedivakova,Kendy Hoang,Jeremiah Tsyporin,Daniel S. Gareau,Mariella G. Filbin,Mariella G. Filbin,Serguei Bannykh,Chintda Santiskulvong,Yizhou Wang,Jie Tang,Mario L. Suvà,Mario L. Suvà,Bin Chen,Moise Danielpour,Joshua J. Breunig +30 more
TL;DR: MADR is established, which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci and provides a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells.
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CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool
Fayu Yang,Changbao Liu,Ding Chen,Mengjun Tu,Haihua Xie,Huihui Sun,Xianglian Ge,Lianchao Tang,Jin Li,Jiayong Zheng,Zongming Song,Jia Qu,Feng Gu +12 more
TL;DR: This work successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time.
Book ChapterDOI
CRISPR/Cas9 for Sickle Cell Disease: Applications, Future Possibilities, and Challenges.
TL;DR: Growth-based approaches to correct the underlying mutation in patient-derived hematopoietic stem/progenitor cells, induce fetal hemoglobin expression to circumvent sickling of red blood cells, or create corrected induced pluripotent stem cells (iPSCs) among other approaches are summarized.
Journal ArticleDOI
Rare Opportunities: CRISPR/Cas-Based Therapy Development for Rare Genetic Diseases
TL;DR: The most significant recent achievements and remaining hurdles in the application of CRISPR/Cas technology to rare diseases are summarized and a glimpse is taken at the exciting road ahead.
References
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Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,Le Cong,F. Ann Ran,F. Ann Ran,David M. Cox,David M. Cox,Shuailiang Lin,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +15 more
TL;DR: The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage as discussed by the authors.
Multiplex Genome Engineering Using CRISPR/Cas Systems
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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