Delivery Approaches for CRISPR/Cas9 Therapeutics in Vivo: Advances and Challenges

Overview

Journal Expert Opin Drug Deliv

Publisher Informa Healthcare

Specialty Pharmacology

Date 2018 Sep 1

PMID 30169977

Citations 60

Authors

D C Luther

Y W Lee

H Nagaraj

F Scaletti

V M Rotello

Affiliations

Soon will be listed here.

Abstract

Introduction: Therapeutic gene editing is becoming a viable biomedical tool with the emergence of the CRISPR/Cas9 system. CRISPR-based technologies have promise as a therapeutic platform for many human genetic diseases previously considered untreatable, providing a flexible approach to high-fidelity gene editing. For many diseases, such as sickle-cell disease and beta thalassemia, curative therapy may already be on the horizon, with CRISPR-based clinical trials slated for the next few years. Translation of CRISPR-based therapy to in vivo application however, is no small feat, and major hurdles remain for efficacious use of the CRISPR/Cas9 system in clinical contexts.

Areas Covered: In this topical review, we highlight recent advances to in vivo delivery of the CRISPR/Cas9 system using various packaging formats, including viral, mRNA, plasmid, and protein-based approaches. We also discuss some of the barriers which have yet to be overcome for successful translation of this technology.

Expert Opinion: This review focuses on the challenges to efficacy for various delivery formats, with specific emphasis on overcoming these challenges through the development of carrier vehicles for transient approaches to CRISPR/Cas9 delivery in vivo.

Citing Articles

Strategic base modifications refine RNA function and reduce CRISPR-Cas9 off-targets.

Zhang K, Shen W, Zhao Y, Xu X, Liu X, Qi Q Nucleic Acids Res. 2025; 53(4).

PMID: 39964477 PMC: 11833691. DOI: 10.1093/nar/gkaf082.

CRISPR-Cas9 Gene Therapy: Non-Viral Delivery and Stimuli-Responsive Nanoformulations.

Lee H, Rho W, Kim Y, Chang H, Jun B Molecules. 2025; 30(3).

PMID: 39942646 PMC: 11820414. DOI: 10.3390/molecules30030542.

Anionic polymer coating for enhanced delivery of Cas9 mRNA and sgRNA nanoplexes.

Chen S, Pinto Carneiro S, Merkel O Biomater Sci. 2024; 13(3):659-676.

PMID: 39687993 PMC: 11650648. DOI: 10.1039/d4bm01290a.

Nucleic acid drugs: recent progress and future perspectives.

Sun X, Setrerrahmane S, Li C, Hu J, Xu H Signal Transduct Target Ther. 2024; 9(1):316.

PMID: 39609384 PMC: 11604671. DOI: 10.1038/s41392-024-02035-4.

Characterization of NiCas12b for In Vivo Genome Editing.

Zhang Y, Wei J, Wang H, Wang Y Adv Sci (Weinh). 2024; 11(36):e2400469.

PMID: 39076074 PMC: 11423069. DOI: 10.1002/advs.202400469.

References

Ding Q, Strong A, Patel K, Ng S, Gosis B, Regan S . Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ Res. 2014; 115(5):488-92. PMC: 4134749. DOI: 10.1161/CIRCRESAHA.115.304351. View

Kozarsky K, Wilson J . Gene therapy: adenovirus vectors. Curr Opin Genet Dev. 1993; 3(3):499-503. DOI: 10.1016/0959-437x(93)90126-a. View

Tabebordbar M, Zhu K, Cheng J, Chew W, Widrick J, Yan W . In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2016; 351(6271):407-411. PMC: 4924477. DOI: 10.1126/science.aad5177. View

Nelson C, Gersbach C . Engineering Delivery Vehicles for Genome Editing. Annu Rev Chem Biomol Eng. 2016; 7:637-62. DOI: 10.1146/annurev-chembioeng-080615-034711. View

Zhou X, Xin J, Fan N, Zou Q, Huang J, Ouyang Z . Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cell Mol Life Sci. 2014; 72(6):1175-84. PMC: 11113635. DOI: 10.1007/s00018-014-1744-7. View

Kim H, Kim J . A guide to genome engineering with programmable nucleases. Nat Rev Genet. 2014; 15(5):321-34. DOI: 10.1038/nrg3686. View

Aubrey B, Kelly G, Kueh A, Brennan M, OConnor L, Milla L . An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo. Cell Rep. 2015; 10(8):1422-32. DOI: 10.1016/j.celrep.2015.02.002. View

Paix A, Folkmann A, Rasoloson D, Seydoux G . High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. Genetics. 2015; 201(1):47-54. PMC: 4566275. DOI: 10.1534/genetics.115.179382. View

Maeder M, Linder S, Cascio V, Fu Y, Ho Q, Joung J . CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013; 10(10):977-9. PMC: 3794058. DOI: 10.1038/nmeth.2598. View

10.

Cradick T, Fine E, Antico C, Bao G . CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 2013; 41(20):9584-92. PMC: 3814385. DOI: 10.1093/nar/gkt714. View

11.

Ehrke-Schulz E, Schiwon M, Leitner T, David S, Bergmann T, Liu J . CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes. Sci Rep. 2017; 7(1):17113. PMC: 5719366. DOI: 10.1038/s41598-017-17180-w. View

12.

Knight S, Xie L, Deng W, Guglielmi B, Witkowsky L, Bosanac L . Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science. 2015; 350(6262):823-6. DOI: 10.1126/science.aac6572. View

13.

Ran F, Cong L, Yan W, Scott D, Gootenberg J, Kriz A . In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520(7546):186-91. PMC: 4393360. DOI: 10.1038/nature14299. View

14.

Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi N . CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature. 2014; 514(7522):380-4. PMC: 4199937. DOI: 10.1038/nature13589. View

15.

Dow L . Modeling Disease In Vivo With CRISPR/Cas9. Trends Mol Med. 2015; 21(10):609-621. PMC: 4592741. DOI: 10.1016/j.molmed.2015.07.006. View

16.

Kaminski R, Chen Y, Fischer T, Tedaldi E, Napoli A, Zhang Y . Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing. Sci Rep. 2016; 6:22555. PMC: 4778041. DOI: 10.1038/srep22555. View

17.

Konermann S, Brigham M, Trevino A, Joung J, Abudayyeh O, Barcena C . Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2014; 517(7536):583-8. PMC: 4420636. DOI: 10.1038/nature14136. View

18.

Shalem O, Sanjana N, Hartenian E, Shi X, Scott D, Mikkelson T . Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2013; 343(6166):84-87. PMC: 4089965. DOI: 10.1126/science.1247005. View

19.

Sternberg S, Doudna J . Expanding the Biologist's Toolkit with CRISPR-Cas9. Mol Cell. 2015; 58(4):568-74. DOI: 10.1016/j.molcel.2015.02.032. View

20.

Hilton I, DIppolito A, Vockley C, Thakore P, Crawford G, Reddy T . Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol. 2015; 33(5):510-7. PMC: 4430400. DOI: 10.1038/nbt.3199. View