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Curated Papers > Highly Cited Authoritative Literature on "CRISPR Gene Editing"

Search-and-replace genome editing without double-strand breaks or donor DNA. ​

Literature Information ​

DOI10.1038/s41586-019-1711-4
PMID31634902
JournalNature
Impact Factor48.5
JCR QuartileQ1
Publication Year2019
Times Cited2053
KeywordsGenome Editing, Prime Editing, Genetic Variants
Literature TypeJournal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.
ISSN0028-0836
Pages149-157
Issue576(7785)
AuthorsAndrew V Anzalone, Peyton B Randolph, Jessie R Davis, Alexander A Sousa, Luke W Koblan, Jonathan M Levy, Peter J Chen, Christopher Wilson, Gregory A Newby, Aditya Raguram, David R Liu

TL;DR ​

This study presents prime editing, a precise genome editing technique that allows for the direct insertion of new genetic information at targeted DNA sites, using a modified Cas9 and engineered reverse transcriptase. The method demonstrated high efficiency in correcting genetic mutations associated with diseases like sickle cell and Tay-Sachs, suggesting it could potentially address up to 89% of known disease-related genetic variants with minimal byproducts and lower off-target effects compared to traditional methods.

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Genome Editing · Prime Editing · Genetic Variants

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.

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Primary Questions Addressed ​

  1. How does prime editing compare to traditional CRISPR methods in terms of efficiency and specificity for correcting genetic diseases?
  2. What are the potential applications of prime editing beyond correcting genetic diseases, such as in agricultural biotechnology?
  3. Can prime editing be used effectively in vivo, and what challenges might arise in delivering the editing components to target tissues?
  4. What are the implications of prime editing's lower off-target effects for therapeutic applications in humans?
  5. How does the design of pegRNA influence the success rate and accuracy of prime editing in various cell types?

Key Findings ​

Research Background and Purpose ​

The study presents "prime editing," a novel genome editing technique aimed at precisely modifying genetic sequences without inducing double-strand breaks (DSBs) or requiring donor DNA templates. Traditional methods, such as CRISPR-Cas9, often lead to unwanted byproducts and are less efficient in correcting a significant portion of pathogenic genetic variants. The primary goal of this research is to develop a method that allows for versatile and accurate genome editing, thereby addressing the challenges faced in correcting the over 75,000 known human genetic variants associated with diseases.

Main Methods/Materials/Experimental Design ​

Prime editing employs a catalytically impaired Cas9 fused to a reverse transcriptase (RT) and guided by a prime editing guide RNA (pegRNA). The pegRNA specifies the target site and encodes the desired edit. The editing process can be summarized as follows:

Mermaid diagram

  1. Components:

    • Cas9 Nickase: A modified Cas9 that creates a single-strand break at the target site.
    • Reverse Transcriptase: An enzyme that synthesizes DNA from an RNA template.
    • pegRNA: Contains the target sequence and the desired edit.

  2. Editing Strategies:

    • PE1: Initial version using wild-type M-MLV RT.
    • PE2: Improved version with engineered RT variants for enhanced efficiency.
    • PE3: Further refinement that includes nicking the non-edited strand to increase editing efficiency.

  3. Cell Types Tested: Human HEK293T cells, K562 cells, U2OS cells, and primary mouse cortical neurons.

Key Results and Findings ​

  • Editing Efficiency: Prime editing showed high efficiency (20-50%) with minimal byproducts (1-10% indels) across various cell types.
  • Types of Edits: The technique successfully performed all 12 types of point mutations, targeted insertions, and deletions.
  • Disease Models: Successfully corrected mutations responsible for sickle cell disease and Tay-Sachs disease in HEK293T cells.
  • Comparison with Other Techniques: Prime editing demonstrated fewer off-target effects and lower byproduct generation compared to traditional CRISPR-Cas9 methods and homology-directed repair (HDR).

Main Conclusions/Significance/Innovation ​

Prime editing represents a significant advancement in genome editing technology, allowing for precise modifications without the drawbacks of DSBs. It expands the range of genetic variants that can be targeted and corrected, potentially addressing 89% of pathogenic variants documented in databases like ClinVar. The flexibility of pegRNA design enables tailored applications in various therapeutic contexts.

Research Limitations and Future Directions ​

  • Efficiency Variability: Editing efficiencies varied across different cell types, suggesting a need for optimization in non-mitotic cells.
  • Off-Target Analysis: While off-target effects were significantly lower than those associated with Cas9, comprehensive genome-wide assessments are necessary.
  • Clinical Applications: Future studies should focus on in vivo applications and integration with delivery systems for therapeutic use.

The study underscores the potential of prime editing to revolutionize genetic medicine, offering a tool for correcting genetic disorders with unprecedented precision and flexibility. Further exploration into its applications and improvements will be crucial for translating this technology into clinical settings.

References ​

  1. In vitro enzymology of Cas9. - Carolin Anders;Martin Jinek - Methods in enzymology (2014)
  2. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. - Benjamin P Kleinstiver;Vikram Pattanayak;Michelle S Prew;Shengdar Q Tsai;Nhu T Nguyen;Zongli Zheng;J Keith Joung - Nature (2016)
  3. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. - Alexis C Komor;Yongjoo B Kim;Michael S Packer;John A Zuris;David R Liu - Nature (2016)
  4. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. - Robert J Ihry;Kathleen A Worringer;Max R Salick;Elizabeth Frias;Daniel Ho;Kraig Theriault;Sravya Kommineni;Julie Chen;Marie Sondey;Chaoyang Ye;Ranjit Randhawa;Tripti Kulkarni;Zinger Yang;Gregory McAllister;Carsten Russ;John Reece-Hoyes;William Forrester;Gregory R Hoffman;Ricardo Dolmetsch;Ajamete Kaykas - Nature medicine (2018)
  5. Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches. - Andrew V Anzalone;Annie J Lin;Sakellarios Zairis;Raul Rabadan;Virginia W Cornish - Nature methods (2016)
  6. ClinVar: public archive of interpretations of clinically relevant variants. - Melissa J Landrum;Jennifer M Lee;Mark Benson;Garth Brown;Chen Chao;Shanmuga Chitipiralla;Baoshan Gu;Jennifer Hart;Douglas Hoffman;Jeffrey Hoover;Wonhee Jang;Kenneth Katz;Michael Ovetsky;George Riley;Amanjeev Sethi;Ray Tully;Ricardo Villamarin-Salomon;Wendy Rubinstein;Donna R Maglott - Nucleic acids research (2016)
  7. 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 - Nucleic acids research (2015)
  8. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. - P Rouet;F Smih;M Jasin - Proceedings of the National Academy of Sciences of the United States of America (1994)
  9. Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. - S Kügler;E Kilic;M Bähr - Gene therapy (2003)
  10. Continuous evolution of base editors with expanded target compatibility and improved activity. - B W Thuronyi;Luke W Koblan;Jonathan M Levy;Wei-Hsi Yeh;Christine Zheng;Gregory A Newby;Christopher Wilson;Mantu Bhaumik;Olga Shubina-Oleinik;Jeffrey R Holt;David R Liu - Nature biotechnology (2019)

Literatures Citing This Work ​

  1. Super-precise new CRISPR tool could tackle a plethora of genetic diseases. - Heidi Ledford - Nature (2019)
  2. Got mutation? 'Base editors' fix genomes one nucleotide at a time. - Sandeep Ravindran - Nature (2019)
  3. A heterodimer of evolved designer-recombinases precisely excises a human genomic DNA locus. - Felix Lansing;Maciej Paszkowski-Rogacz;Lukas Theo Schmitt;Paul Martin Schneider;Teresa Rojo Romanos;Jan Sonntag;Frank Buchholz - Nucleic acids research (2020)
  4. The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements. - Petros Patsali;Claudio Mussolino;Petros Ladas;Argyro Floga;Annita Kolnagou;Soteroula Christou;Maria Sitarou;Michael N Antoniou;Toni Cathomen;Carsten Werner Lederer;Marina Kleanthous - Journal of clinical medicine (2019)
  5. Advances in Sphingolipidoses: CRISPR-Cas9 Editing as an Option for Modelling and Therapy. - Renato Santos;Olga Amaral - International journal of molecular sciences (2019)
  6. Human germline genome editing. - Rebecca A Lea;Kathy K Niakan - Nature cell biology (2019)
  7. Advances in genome editing through control of DNA repair pathways. - Charles D Yeh;Christopher D Richardson;Jacob E Corn - Nature cell biology (2019)
  8. Interplay between MicroRNAs and Oxidative Stress in Neurodegenerative Diseases. - Julia Konovalova;Dmytro Gerasymchuk;Ilmari Parkkinen;Piotr Chmielarz;Andrii Domanskyi - International journal of molecular sciences (2019)
  9. Evolutionary Dynamics of Structural Variation at a Key Locus for Color Pattern Diversification in Cichlid Fishes. - Claudius F Kratochwil;Yipeng Liang;Sabine Urban;Julián Torres-Dowdall;Axel Meyer - Genome biology and evolution (2019)
  10. SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing. - Chiao-Lin Chen;Jonathan Rodiger;Verena Chung;Raghuvir Viswanatha;Stephanie E Mohr;Yanhui Hu;Norbert Perrimon - G3 (Bethesda, Md.) (2020)

... (2043 more literatures)

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