CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors

Abstract

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments.

Figure 1.. Therapeutic editing strategies based on nuclease activity.

(a) Gene disruption introduces indel mutations (red and orange bars) into a gene (dark gray bars), silencing the gene function, (b) Targeting can be specific to the mutant allele (red bar) and spare the normal allele, (c) HDR mediates precise mutation repair in the presence of a donor template, (d) Targeted insertion of a therapeutic gene (green bar) can be achieved by either HDR or HITI. (e) Two cuts can delete a large gene fragment harboring a mutation, resulting in the production of truncated by partially functional protein, (f) Allelic exchange converts a compound heterozygous genotype to heterozygous through translation between two homologous chromosomes (dashed lines). The targeting site is designed to be intronic, so that the indel mutations caused by DNA repair (yellow bars) do not compromise gene expression, (g) RNA targeting achieves gene silencing by degrading RNA instead of DNA. NHEJ: non-homologous end joining. HDR: homology-directed repair. HITI: homology-independent targeted integration.

Figure 2.. Cas-effector fusion platforms for therapeutic purposes.

(a) Cas protein (gray shape) fused a transcriptional inhibitor (red oval) or activator (green oval) can repress or boost transcription, respectively, (b) Cas protein fused with an enzyme that can modify epigenetic marks of DNA (blue flags) or histone (brown flag) is useful to change gene expression status, (c) A DNA base editor consists of a dCas or nCas protein, and an adenine or cytosine deaminase (orange oval) that converts A to G or C to T, respectively (red bars to green bars). Note that the editing window (orange shade) may contain by-stander editing targets. An RNA base editor can mediate A to I or C to U base change (red diamond to green diamond), (d) Prime editor comprises an nCas protein fused with a reverse transcriptase (purple oval). Note that the editing window (purple shade) starts from 3 base pairs upstream of the PAM (red bars) of SpCas9, and the editing outcome can be diverse (rainbow shade), including all single base changes and small indels (not shown).

Figure 3.. Reconstitution of full-length protein expression from two AAV vectors.

(a) The two AAV vector genomes can undergo recombination mediated by an overlapping sequence (1), ITR (2), or a recombinogenic sequence (3) to form a recombined vector DNA that encodes the full-length protein. The transcript derived from the recombined vector DNA may undergo splicing by engineered splicing signals (purple pentagons), (b) The two mRNAs transcribed from individual vector genomes can undergo trans-splicing to form a chimeric mRNA encoding the full-length protein, which is promoted by the hybridization domains (short black bars), (c) The two half proteins expressed from the two AAV vectors can undergo protein trans-splicing mediated by split inteins (diamonds) to form a full-length protein.