Next-Generation CRISPR Technologies and Their Applications in Gene and Cell Therapy

Abstract

The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) nucleases has transformed biotechnology by providing an easy, efficient, and versatile platform for editing DNA. However, traditional CRISPR-based technologies initiate editing by activating DNA double-strand break (DSB) repair pathways, which can cause adverse effects in cells and restrict certain therapeutic applications of the technology. To this end, several new CRISPR-based modalities have been developed that are capable of catalyzing editing without the requirement for a DSB. Here, we review three of these technologies: base editors, prime editors, and RNA-targeting CRISPR-associated protein (Cas)13 effectors. We discuss their strengths compared to traditional gene-modifying systems, we highlight their emerging therapeutic applications, and we examine challenges facing their safe and effective clinical implementation.

Keywords: CRISPR; CRISPR-Cas13; base editing; gene therapy; prime editing.

Figure 1.. Overview of next-generation DNA editing technologies.

(A) Fourth generation CBEs (BE4) consist of fusions of nCas9 with a cytosine deaminase domain (APOBEC1) that catalyzes the deamination of cytosine and two uracil DNA glycosylase inhibitor (UGI) domains, which can prevent the unwanted excision of uracil by endogenous uracil N-glycosylase enzymes. To catalyze base editing, CBEs are directed to a specific DNA sequence via an sgRNA. Then, following nCas9 binding and denaturation of the target DNA sequence, the APOBEC1 enzyme binds to cytosines within a stretch of sequence in the exposed DNA strand and catalyzes a deamination reaction that results in the conversion of cytosine to uracil, which is recognized by cells as thymine during replication and/or repair. (B) ABE7.10 catalyzes A > G transitions and consists of a tandem fusion of a wild type tRNA adenosine deaminase (TadA) and an evolved TadA domain, engineered to accept DNA as a substrate, fused with nCas9. Deamination of adenosine yields inosine, which is recognized by cells as guanosine. (C) Therapeutic outcomes of base editing include: (1) the correction of single point mutations that encode for pathogenic proteins, (2) the introduction of premature stop codons to eliminate pathogenic transcripts via the nonsense-mediated decay (NMD) surveillance pathway, and (3) regulation of mRNA splicing by editing splicing motifs – SA and SD – to induce exon skipping. (D) Prime editors (PEs) consist of a prime editing guide RNA (pegRNA), which both specifies the target sequence and encodes the desired edits, and nCas9 fused to a reverse transcriptase (RT) domain, which promotes the incorporation of the edit encoded in the pegRNA template into the target DNA sequence. The RT fusion provides high levels of design versatility for achieving a range of editing outcomes. (E) Examples of therapeutic prime editing outcomes include: (1) the correction of single or multiple mutations that result in pathogenic proteins and (2) the insertion or deletion of pathogenic sequences. Additional abbreviations are as follows: SA, splice acceptor site; SD, splice donor site.

Figure 2.. Methods of reconstituting large gene-editing proteins delivered by dual AAV vectors.

(A) ITR recombination can mediate the joining of two ITR-flanked viral genomes that each encode a portion of a base editor sequence. In the case of an ABE, the first vector encodes: (1) an sgRNA driven by the U6 promoter, (2) a promoter to drive expression of the NLS-tagged tandem TadA domains fused to the N-terminal (NT) half of a split nCas9 transgene and a splice donor (SD) sequence, while the second vector carries: (1) a splice acceptor (SA) signal sequence, (2) the C-terminal (CT) half of the split nCas9 fused to an NLS tag and an HA epitope tag and (3) a bGH polyadenylation (polyA) signal sequence. Once in the target cell, the two viral vectors are joined by a recombination reaction between the two ITRs and the full-length ABE pre-mRNA is transcribed. pre-mRNA processing removes the intron and the intervening ITR, resulting in the translation of a full-length ABE protein. (B) Intein-mediated protein trans-splicing of two separately expressed base editor halves can be used to assemble a full-length base editor protein. In the case of a CBE, the first vector encodes: (1) a promoter to drive expression of the NT of the base editor, (2) a V5 epitope and NLS tag followed by the APOBEC1 domain fused to the NT fragment of the split nCas9 half and a connected N-terminal intein domain, and (3) a bGH poly A signal sequence, while the second vector carries: (1) a promoter to drive the expression of the C-terminal intein domain fused to the CT half of nCas9 linked to two UGI domains, an NLS and an HA epitope tag, and (3) a poly A signal sequence. This vector also encodes an sgRNA expression cassette. Both vectors are packaged and co-delivered in vivo, where, once internalized by the cells, the separate intein-split base editor protein halves are expressed. Following their translation, the N and C intein fragments associate and catalyze a trans-splicing reaction that removes the intein moieties and results in the formation of the full-length CBE. Additional abbreviations are as follows: TadA, tRNA adenosine deaminase; NLS, nuclear localization signal; ITR, inverted terminal repeat; bGH, bovine growth hormone; APOBEC1, cytosine deaminase domain.

Figure 3.. Overview of RNA-targeting CRISPR systems.

(A, left) To catalyze RNA cleavage, a CRISPR RNA (crRNA) molecule binds to Cas13 and directs it to a target RNA site, which stimulates the RNase catalytic activity of the HEPN domains of Cas13, triggering its cleavage. Cas13 systems can also mediate targeted splicing utilizing dCas13 and a single or multiple crRNA molecules that bind to splice site motifs – SA and SD – and likely block them from recognition by the splicing machinery. (A, right) Additionally, Cas13 effectors can serve as scaffolds to enable targeted RNA editing. RNA editors consist of fusions of dCas13 to adenosine deaminases acting on RNA (ADAR) to stimulate A to I edits and cytidine deaminase domains to mediate C to U conversions. (B) Examples of therapeutic RNA targeting outcomes include the use of Cas13 protein to cleave pathogenic transcripts to prevent the translation of mutated proteins and the use of dCas13 to bind splice donor and acceptor sites to block spliceosome protein complexes from triggering exon inclusion that may encode for pathogenic isoforms. Additionally, RNA editing can be used to correct single-point mutations in mRNA that encode for pathogenic proteins. Abbreviations as follows: E1, exon 1; E2, exon2; E3, exon 3; SA, splice acceptor site; SD, splice donor site.