CRISPR/Cas9 therapeutics: progress and prospects

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. A variety of efficient Cas9 variants and derivatives have been developed to cope with the complex genomic changes that occur during diseases. However, strategies to effectively deliver the CRISPR system to diseased cells in vivo are currently lacking, and nonviral vectors with target recognition functions may be the focus of future research. Pathological and physiological changes resulting from disease onset are expected to serve as identifying factors for targeted delivery or targets for gene editing. Diseases are both varied and complex, and the choice of appropriate gene-editing methods and delivery vectors for different diseases is important. Meanwhile, there are still many potential challenges identified when targeting delivery of CRISPR/Cas9 technology for disease treatment. This paper reviews the current developments in three aspects, namely, gene-editing type, delivery vector, and disease characteristics. Additionally, this paper summarizes successful examples of clinical trials and finally describes possible problems associated with current CRISPR applications.

Fig. 1

Timeline of major events in the development of CRISPR/Cas technology and representative Cas9 variants. In 1987, the CRISPR sequence was first reported. The mechanism by which Cas9 cuts DNA double strands was reported in 2012, and Cas9 was subsequently used for gene editing in mammalian cells. Since then, CRISPR technology has developed rapidly, and multiple Cas9 variants with specific functions have been identified. The representative variants are single-base substitution tools (e.g., CBE and PE) and transcriptional regulatory tools (e.g., dCas9-effector). Since 2016, CRISPR-based gene-editing technologies have been successively used in clinical treatment with great success. CRISPR clustered regularly interspaced short palindromic repeats, Cas CRISPR-associated, dCas9 dead Cas9, PAM protospacer-adjacent motifs, CBE cytosine base editors, ABE adenine base editors, GBE glycosylase base editors. (Figure was created with Adobe Illustrator)

Fig. 2

Schematic diagram of DNA strand cleavage tools. a Cas9 cleaves DNA double strands to form flat ends. b Cas9 nickase (Cas9n) cleaves the single DNA strand. c Cas12a cuts DNA double strands to form sticky ends. d Cas13a recognizes and cleaves RNA strands. (Figure was created with Biorender.com)

Fig. 3

Schematic diagram of dCas9-based tools to regulate expression. a The dCas9 fusion VP64, VPR and other transcriptional activation effectors bind near the gene transcription start site to promote gene transcription. b dCas9 may be fused with KRAB or other transcriptional repressor effectors and bind to the gene transcription start site to silence gene transcription. c The complex formed by the fusion of dCas9 with P300 or other histone acetylases binds the gene transcription start site or enhancer region and promotes histone acetylation, which in turn enhances gene transcription. d dCas9 fused with DNMT3 and other DNA methyltransferases may bind the gene transcription start site to promote DNA methylation and thereby knock down gene transcription. (Figure was created with Biorender.com)

Fig. 4

Schematic diagram of the single-base substitution tool. a Fusion of Cas9n with adenosine deaminase or cytidine deaminase enables the introduction of point mutations in the genome, APOBEC1 induces a C to U mutation, and TadA induces an A to I mutation. b PE contains a 30 bp segment of pegRNA, including the PBS sequence and RT region. PBS binds to the DNA strand and synthesizes the complementary strand of the RT region in the presence of reverse transcriptase. PBS primer binding site, RT reverse transcriptase, pegRNA prime editing guide RNA. (Figure was created with Biorender.com)

Fig. 5

Schematic diagram showing multiple types of vectors for the in vivo delivery of CRISPR systems. The central region shows three forms of CRISPR action: pDNA, mRNA, and RNP. The middle circle section shows examples of delivery carriers, and the outermost area shows how the carriers are produced or the components. SU surface envelope protein, TM transmembrane envelope protein. (Figure was created with Adobe Illustrator and Biorender.com)

Fig. 6

Delivering the CRISPR/Cas9 system to treat cancer, liver fibrosis, obesity, and cardiovascular diseases. a APACPs, exosomes, PICASSO and CHO-PEGA were intravenously injected into mice, and AAV9 was intraperitoneally injected. b APACPs are transported through the blood circulation to the tumor tissue, and hypoxic conditions promote the entry of APACPs into tumor cells. NPs release RNPs, silence the expression of HSP90α and reduce the hyperthermia tolerance of tumor cells. Externally applied NIR-induced photothermal therapy kills tumor cells. PICASSO responds to MMP-2 on the tumor cell membrane, and the shell disintegrates and the core enters the cell by endocytosis. The plasmid escapes from the lysosome into the cytoplasm through the proton sponge effect. c AAV9 delivered sgRNAs targeting Mef2d and Klf15 into dCas9-VPR transgenic mice. dCas9-VPR was synergistically transcribed with Myh6 and therefore specifically activated the expression of Mef2d and Klf15 in cardiomyocytes. Lipid nanoparticles CHO-PEGA deliver CRISPR/Cas9 to vascular smooth muscle cells in aortic coarctation to knockdown Fbn1. d Adipocyte targeting sequence to 9-mer arginine (ATS-9R) recognizes forbidden elements expressed at high levels in adipose tissue and delivers plasmids into white adipocytes, which contain huge lipid droplets and large amounts of triglycerides and cholesterol and release large amounts of inflammatory factors. After interfering with the expression of fatty acid binding protein 4 (Fabp4), the size of lipid droplets in white adipocytes decreases, and the release of inflammatory factors is inhibited for the purpose of treating obesity. e The exosomes secreted by LX-2 cells were extracted, and RNPs were loaded into the exosomes by electroporation. In studies targeting the knockdown of PUMA, CcnE1, and KAT5, exosomes were effective at alleviating liver diseases such as liver fibrosis. In vitro transfection of plasmids encoding sgRNA and dCas9/VP64 into mouse liver AML12 cells resulted in the secretion of AML12 exosomes carrying the CRISPR/dCas9 system. Delivery of these exosomes to HSCs elevated HNF4α expression and prompted cell differentiation into hepatocytes. (Figure was created with Adobe Illustrator and Biorender.com)

Fig. 7

Summary chart of FDA-approved CRISPR therapies that can be used in clinical treatments. The text includes the date of FDA approval, the name of the therapy, and the type of applied diseases. DMD Duchenne muscular dystrophy, SCD sickle cell disease, TDT transfusion-dependent β-thalassemia, LCA10 Leber congenital amaurosis type 10, TTR transthyretin. (Figure was created with Biorender.com)