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

Han Zhang; Karen Kelly; Jonathan Lee; Dimas Echeverria; David Cooper; Rebecca Panwala; Nadia Amrani; Zexiang Chen; Nicholas Gaston; Atish Wagh; Gregory A Newby; Jun Xie; David R Liu; Guangping Gao; Scot A Wolfe; Anastasia Khvorova; Jonathan K Watts; Erik J Sontheimer

doi:10.1093/nar/gkad1125

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

Nucleic Acids Res. 2024 Jan 25;52(2):977-997. doi: 10.1093/nar/gkad1125.

Authors

Han Zhang¹, Karen Kelly¹, Jonathan Lee¹, Dimas Echeverria¹, David Cooper¹, Rebecca Panwala¹, Nadia Amrani¹, Zexiang Chen¹, Nicholas Gaston¹, Atish Wagh¹, Gregory A Newby^{2

3

4}, Jun Xie^{5

6

7

8}, David R Liu^{2

3

4}, Guangping Gao^{5

6

7

8}, Scot A Wolfe^{8

9}, Anastasia Khvorova^{1

10

11}, Jonathan K Watts^{1

12

10

8}, Erik J Sontheimer^{1

11

8}

Affiliations

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

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.

MeSH terms

Animals
Gene Editing*
Mice
Oligonucleotides
RNA / genetics
RNA, Guide, CRISPR-Cas Systems*
Tissue Distribution

Substances

RNA, Guide, CRISPR-Cas Systems
RNA
Oligonucleotides

Abstract

MeSH terms

Substances

Grants and funding