Search-and-replace genome editing without double-strand breaks or donor DNA
Andrew V. Anzalone,Andrew V. Anzalone,Andrew V. Anzalone,Peyton B. Randolph,Peyton B. Randolph,Peyton B. Randolph,Jessie Rose Davis,Jessie Rose Davis,Jessie Rose Davis,Alexander A. Sousa,Alexander A. Sousa,Alexander A. Sousa,Luke W. Koblan,Luke W. Koblan,Luke W. Koblan,Jonathan M. Levy,Jonathan M. Levy,Jonathan M. Levy,Peter J. Chen,Peter J. Chen,Peter J. Chen,Christine D. Wilson,Christine D. Wilson,Christine D. Wilson,Gregory A. Newby,Gregory A. Newby,Gregory A. Newby,Aditya Raguram,Aditya Raguram,Aditya Raguram,David R. Liu,David R. Liu,David R. Liu +32 more
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TLDR
A new DNA-editing technique called prime editing offers improved versatility and efficiency with reduced byproducts compared with existing techniques, and shows potential for correcting disease-associated mutations.Abstract:
Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2-5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.read more
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Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity
Ida Höijer,Josefin Johansson,Sanna Gudmundsson,Sanna Gudmundsson,Sanna Gudmundsson,Chen-Shan Chin,Ignas Bunikis,Susana Häggqvist,Anastasia Emmanouilidou,Anastasia Emmanouilidou,Maria Wilbe,M. den Hoed,M. den Hoed,Marie-Louise Bondeson,Lars Feuk,Ulf Gyllensten,Adam Ameur,Adam Ameur +17 more
TL;DR: SMRT-OTS and Nano-OTS, two amplification-free long-read sequencing protocols for detection of gRNA driven digestion of genomic DNA by Cas9, revealed many gRNA binding sites in vitro that would be difficult to predict based on gRNA sequence alignment to a reference.
Journal ArticleDOI
Novel therapies for mucopolysaccharidosis type III
Berna Seker Yilmaz,Berna Seker Yilmaz,James Davison,Simon Jones,Julien Baruteau,Julien Baruteau,Julien Baruteau +6 more
TL;DR: The numerous approaches of curative therapy developed for MPS III are reviewed from historical ineffective haematopoietic stem cell transplantation and substrate reduction therapy to the promising ongoing clinical trials based on enzyme replacement therapy or adeno‐associated or lentiviral vectors mediated gene therapy.
Journal ArticleDOI
The Potential of Genome Editing for Improving Seed Oil Content and Fatty Acid Composition in Oilseed Crops.
Udaya Subedi,Udaya Subedi,Kethmi N. Jayawardhane,Xue Pan,Jocelyn A. Ozga,Guanqun Chen,Nora A. Foroud,Stacy D. Singer +7 more
TL;DR: A number of genes that may provide ideal targets for genome editing in this context are examined, as well as new CRISPR-related tools that have the potential to be applied to oilseed plants and may allow additional gains to be made in the future.
Journal ArticleDOI
Double-Barreled CRISPR Technology as a Novel Treatment Strategy For COVID-19
TL;DR: This Perspective discusses coronavirus pathogenicity, conventional antiviral therapies, prophylactic strategies, and novel treatment strategies for COVID-19, and highlights the application of CRISPR technology as an emerging pan-antiviral therapy.
Journal ArticleDOI
Plant genome editing: ever more precise and wide reaching
TL;DR: A review of recent highlights in the field of precise gene editing, chromosome engineering and genome engineering technology in plants can be found in this article, where the authors introduce and discuss recent highlights.
References
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limma powers differential expression analyses for RNA-sequencing and microarray studies
Matthew E. Ritchie,Belinda Phipson,Di Wu,Yifang Hu,Charity W. Law,Wei Shi,Gordon K. Smyth,Gordon K. Smyth +7 more
TL;DR: The philosophy and design of the limma package is reviewed, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.
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RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome
Bo Li,Colin N. Dewey +1 more
TL;DR: It is shown that accurate gene-level abundance estimates are best obtained with large numbers of short single-end reads, and estimates of the relative frequencies of isoforms within single genes may be improved through the use of paired- end reads, depending on the number of possible splice forms for each gene.
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Martin Jinek,Krzysztof Chylinski,Krzysztof Chylinski,Ines Fonfara,Michael H. Hauer,Jennifer A. Doudna,Emmanuelle Charpentier +6 more
TL;DR: This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
Journal ArticleDOI
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.