Targeted intracellular delivery of Cas13 and Cas9 nucleases using bacterial toxin-based platforms

Cell Reports

Evan Appleton¹  George M. Church²  Yang Liu²  Min Dong³  Songhai Tian⁴  Huan Wang⁴ 

[1] Corresponding author;Department of Microbiology and Department of Surgery, Harvard Medical School, Boston, MA 02115, USA;Department of Nephrology, The First Hospital of Jilin University, Changchun, 130021, China;Department of Urology, Boston Children's Hospital, Boston, MA 02115, USA;

关键词: CRISPR-Cas9;    Cas9;    Cas13;    Cre;    intracellular delivery;    toxin;   

DOI  :  

来源: DOAJ

【 摘 要 】

Summary: Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful. Delivery of Cas13a and CasRx, together with guide RNA expression, reduces mRNAs encoding GFP, SARS-CoV-2 fragments, and endogenous proteins PPIB, KRAS, and CXCR4 in multiple cell lines. Delivery of Cre recombinase modifies the reporter loci in cells. Delivery of Cas9, together with guide RNA expression, generates mutations at the targeted genomic sites in cell lines and induced pluripotent stem cell (iPSC)-derived human neurons. These findings establish modular delivery systems based on single-chain bacterial toxins for delivery of membrane-impermeable therapeutics into targeted cells.

【 授权许可】

Unknown