Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing <i>in vivo</i>-Reference-Cited by-同舟云学术

Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo

Published:2023-11-30 Issue:2 Volume:52 Page:977-997
ISSN:0305-1048
Container-title:Nucleic Acids Research
language:en
Short-container-title:

Author:

Zhang Han¹,Kelly Karen¹,Lee Jonathan¹,Echeverria Dimas¹^ORCID,Cooper David¹,Panwala Rebecca¹,Amrani Nadia¹,Chen Zexiang¹,Gaston Nicholas¹^ORCID,Wagh Atish¹,Newby Gregory A²³⁴,Xie Jun⁵⁶⁷⁸^ORCID,Liu David R²³⁴,Gao Guangping⁵⁶⁷⁸,Wolfe Scot A⁸⁹^ORCID,Khvorova Anastasia¹¹⁰¹¹^ORCID,Watts Jonathan K¹¹²¹⁰⁸^ORCID,Sontheimer Erik J¹¹¹⁸^ORCID

Affiliation:

1. RNA Therapeutics Institute, University of Massachusetts Chan Medical School , Worcester , MA 01605 , USA

2. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT , Cambridge , MA 02142 , USA

3. Department of Chemistry and Chemical Biology, Harvard University , Cambridge , MA 02139 , USA

4. Howard Hughes Medical Institute, Harvard University , Cambridge , MA 02139 , USA

5. Horae Gene Therapy Center, University of Massachusetts Chan Medical School , Worcester , MA 01605 , USA

6. Viral Vector Core, University of Massachusetts Chan Medical, School , Worcester , MA 01605 , USA

7. Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School , Worcester , MA 01605 , USA

8. Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School , Worcester , MA 01605 , USA

9. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School , Worcester , MA 01605 , USA

10. NeuroNexus Institute, University of Massachusetts Chan Medical School , Worcester , MA 01605 , USA

11. Program in Molecular Medicine, University of Massachusetts Chan Medical School , Worcester , MA 01605 , USA

12. Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School , Worcester , MA 01605 , USA

Abstract

Abstract Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a ‘protecting oligo’), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

Funder

National Institutes of Health

National Center for Advancing Translational Sciences

NIAID

SCGE Collaborative Opportunity Fund

UMass Chan Flow Cytometry Core

UMass Chan Nucleic Acid Chemistry Center

Publisher

Oxford University Press (OUP)

Subject

Genetics

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