Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

Eleonora I. Ioannidi, Matthew T. N. Yarnall, Cian Schmitt-Ulms, Rohan N. Krajeski, Justin Lim, Lukas Villiger, Wenyuan Zhou, Kaiyi Jiang, Nathaniel Roberts, Liyang Zhang, Christopher A. Vakulskas, John A. Walker II, Anastasia P. Kadina, Adrianna E. Zepeda, Kevin Holden, Jonathan S. Gootenberg, Omar O. Abudayyeh
doi: https://doi.org/10.1101/2021.11.01.466786
Eleonora I. Ioannidi
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
2ETH Zürich, Rämistrasse 101, 8092 Zürich, Switzerland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Matthew T. N. Yarnall
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Cian Schmitt-Ulms
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rohan N. Krajeski
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Justin Lim
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lukas Villiger
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Wenyuan Zhou
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kaiyi Jiang
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nathaniel Roberts
3Integrated DNA Technologies, 1710 Commercial Park, Coralville, Iowa 52241, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Liyang Zhang
3Integrated DNA Technologies, 1710 Commercial Park, Coralville, Iowa 52241, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Christopher A. Vakulskas
3Integrated DNA Technologies, 1710 Commercial Park, Coralville, Iowa 52241, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
John A. Walker II
4Synthego Corporation, 3696 Haven Avenue, Suite A, Redwood City, CA 94063
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Anastasia P. Kadina
4Synthego Corporation, 3696 Haven Avenue, Suite A, Redwood City, CA 94063
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Adrianna E. Zepeda
4Synthego Corporation, 3696 Haven Avenue, Suite A, Redwood City, CA 94063
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kevin Holden
4Synthego Corporation, 3696 Haven Avenue, Suite A, Redwood City, CA 94063
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jonathan S. Gootenberg
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: omarabu@mit.edu jgoot@mit.edu
Omar O. Abudayyeh
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: omarabu@mit.edu jgoot@mit.edu
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

Abstract

Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as ∼36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy.

One Sentence Summary A new technology combining CRISPR-mediated genome editing and site-specific integrases enables efficient programmable gene integration at any targeted genomic locus without double-strand DNA breaks, leading to broad applications in basic science research, cell engineering, and gene therapy.

Competing Interest Statement

O.O.A. and J.S.G. are co-inventors on patent applications filed by MIT relating to work in this manuscript. O.O.A. and J.S.G. are co-founders of Sherlock Biosciences, Proof Diagnostics, Moment Biosciences, and Tome Biosciences. O.O.A. and J.S.G. were advisors for Beam Therapeutics during the course of this project.

Footnotes

  • ↵‡ These authors jointly supervised the work.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted November 01, 2021.
Download PDF

Supplementary Material

Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases
Eleonora I. Ioannidi, Matthew T. N. Yarnall, Cian Schmitt-Ulms, Rohan N. Krajeski, Justin Lim, Lukas Villiger, Wenyuan Zhou, Kaiyi Jiang, Nathaniel Roberts, Liyang Zhang, Christopher A. Vakulskas, John A. Walker II, Anastasia P. Kadina, Adrianna E. Zepeda, Kevin Holden, Jonathan S. Gootenberg, Omar O. Abudayyeh
bioRxiv 2021.11.01.466786; doi: https://doi.org/10.1101/2021.11.01.466786
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools
Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases
Eleonora I. Ioannidi, Matthew T. N. Yarnall, Cian Schmitt-Ulms, Rohan N. Krajeski, Justin Lim, Lukas Villiger, Wenyuan Zhou, Kaiyi Jiang, Nathaniel Roberts, Liyang Zhang, Christopher A. Vakulskas, John A. Walker II, Anastasia P. Kadina, Adrianna E. Zepeda, Kevin Holden, Jonathan S. Gootenberg, Omar O. Abudayyeh
bioRxiv 2021.11.01.466786; doi: https://doi.org/10.1101/2021.11.01.466786

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Bioengineering
Subject Areas
All Articles
  • Animal Behavior and Cognition (4397)
  • Biochemistry (9623)
  • Bioengineering (7118)
  • Bioinformatics (24928)
  • Biophysics (12651)
  • Cancer Biology (9984)
  • Cell Biology (14392)
  • Clinical Trials (138)
  • Developmental Biology (7982)
  • Ecology (12141)
  • Epidemiology (2067)
  • Evolutionary Biology (16019)
  • Genetics (10946)
  • Genomics (14772)
  • Immunology (9895)
  • Microbiology (23729)
  • Molecular Biology (9500)
  • Neuroscience (51034)
  • Paleontology (370)
  • Pathology (1544)
  • Pharmacology and Toxicology (2690)
  • Physiology (4035)
  • Plant Biology (8687)
  • Scientific Communication and Education (1512)
  • Synthetic Biology (2403)
  • Systems Biology (6452)
  • Zoology (1349)