Advances in CRISPR therapeutics

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) renaissance was catalysed by the discovery that RNA-guided prokaryotic CRISPR-associated (Cas) proteins can create targeted double-strand breaks in mammalian genomes. This finding led to the development of CRISPR systems that harness natural DNA repair mechanisms to repair deficient genes more easily and precisely than ever before. CRISPR has been used to knock out harmful mutant genes and to fix errors in coding sequences to rescue disease phenotypes in preclinical studies and in several clinical trials. However, most genetic disorders result from combinations of mutations, deletions and duplications in the coding and non-coding regions of the genome and therefore require sophisticated genome engineering strategies beyond simple gene knockout. To overcome this limitation, the toolbox of natural and engineered CRISPR-Cas systems has been dramatically expanded to include diverse tools that function in human cells for precise genome editing and epigenome engineering. The application of CRISPR technology to edit the non-coding genome, modulate gene regulation, make precise genetic changes and target infectious diseases has the potential to lead to curative therapies for many previously untreatable diseases.

Fig. 1. The CRISPR–Cas9 system.

a | CRISPR–Cas9 evolved as a prokaryotic adaptive immune system to protect against phages and other mobile genetic elements. The prokaryotic genome encodes a CRISPR array that contains spacers — short pieces of DNA that have exact homology to the genome of the invading pathogen — separated by repeats. Once transcribed, the array is processed into short CRISPR RNAs (crRNAs), each containing one spacer. The crRNAs duplex with trans-activating CRISPR RNAs (tracrRNAs) to create the secondary structure needed to interact with Cas9 and form a ribonucleoprotein (RNP) complex. In prokaryotes, the Cas9 RNP surveys the cell and binds to the phage genome. Cas9 cuts the phage DNA, creating a double-strand break (DSB) and disrupting the pathogen’s life cycle. b | To import CRISPR–Cas9 into other organisms or cells, the crRNA and tracrRNA are fused into a single guide RNA (sgRNA) that encodes a spacer targeting the genome at a defined site. The sgRNA together with Cas9 can be delivered as DNA via a viral vector or as RNA or protein via a lipid nanoparticle. In mammalian cells, Cas9 RNP creates a DSB and induces DNA repair pathways to generate nucleotide insertions and deletions (indels), leading to a gene edit that can potentially be used to treat disease. AAV, adeno-associated virus.

Fig. 2. Natural Cas systems.

Mining the prokaryotic metagenome has uncovered numerous Cas systems, each with their own unique capabilities that can be leveraged for therapeutic genome engineering. These systems are categorized into two classes: class I systems, which perform their nucleic acid-targeting and nuclease activity as multiple proteins, and class II systems, which have both functions encoded on one protein. a | Type I-E, also known as Cascade, is a class I double-stranded DNA (dsDNA) nuclease. The ability of type I-E to handle longer guide RNAs (gRNAs) means that it can target genomes with higher specificity than other Cas systems. b | Type I-F can interact with TniQ, part of a transposase complex, and is capable of targeted transposition, inserting entirely new DNA sequences into the genome. c | Type II-A, also known as Cas9, is the first CRISPR–Cas system to be used therapeutically in humans. Cas9 can use single guide RNA (sgRNA) and typically has high GC protospacer adjacent motifs (PAMs). d | Type V-A, also known as Cas12a, typically has high AT PAMs and therefore has access to different genomic regions than Cas9. Cas12a can process multiple CRISPR RNAs (crRNAs) from a single transcript, enabling facile multiplexed DNA targeting. e | Type V-F, also known as Cas12f, is an extremely small nuclease (1.4–1.6 kb), which makes it more amenable to viral packaging than other Cas systems. Cas12f has high AT PAMs. f | Type VI-D, also known as Cas13d, targets single-stranded RNA (ssRNA), enabling transcriptome modification, and has no PAM requirement. ssDNA, single-stranded DNA.

Fig. 3. Genome engineering using engineered Cas proteins.

Catalytic residues in Cas proteins can be mutated to render the protein nuclease dead (dCas). Fusing dCas to other protein domains endows novel functionality that can be targeted to precise locations on the human genome. a | dCas fused to transcriptional repressors such as the Krüppel-associated box (KRAB) generates CRISPR interference (CRISPRi), which is capable of targeted gene downregulation lasting for as long as the fusion is present. b | Fusion of dCas to transcriptional activators such as VP64 generates CRISPR activation (CRISPRa), which enables precise gene upregulation while the fusion protein is bound. c | Targeted DNA methylation enabled by fusion of DNA methyltransferases such as DNMT3 to dCas results in long-term and even heritable gene repression that is independent of the fusion protein being bound to the genome. d | Natural or engineered DNA methylation can be removed by fusing methylcytosine dioxygenases such as TET to dCas. e | Other targeted epigenetic marks can be generated by fusing histone methyl or acetyl transferases such as p300 to dCas to modify histone residues leading to long-term, stable upregulation or downregulation of targeted genes. f | Fusing dCas or nickase Cas (nCas), which creates single-stranded DNA breaks, to the cytosine deaminase APOBEC1 converts local cytosines into uracil, which is later converted into thymine^–. g | Fusion of adenosine deaminases such as TadA to dCas or nCas leads to the local conversion of adenosine into inosine, which is resolved as guanine^–. h | Prime editing uses a long prime editing guide RNA (pegRNA) that binds to a nicked DNA strand. This creates the starting conditions for the reverse transcriptase (RT) fused to nCas9 to write the genetic information encoded on the pegRNA directly on the genome. The pegRNA can be designed to enable use of prime editing to create large insertions or deletions.

Fig. 4. Editing non-coding regions of the genome.

CRISPR technologies can be used to edit non-coding regions of the human genome. a | Proper intron splicing can be restored by creating targeted cuts around an intronic mutation (Mut). These cuts lead to deletion or inversion of the mutated region and thereby correct the mRNA^,–. b | Entire exons carrying deleterious mutations can be skipped by modifying the flanking introns^–. c | The expression of a gene can be indirectly increased by knocking out the expression of its inhibitory transcription factor, for example, by indel formation in the enhancer region of the transcription factor^–. d | Single or dual guide RNA (gRNA) strategies that target Cas proteins around a transcriptional start site cause the region to be deleted, leading to a reduction in gene expression^–. e | Editing long non-coding RNA (lncRNA) can modulate gene dosage. For example, in settings in which a lncRNA silences a paternal allele, leaving only a mutant maternal allele, the generation of indels in the lncRNA can disrupt its function, leading to the therapeutic expression of the wild-type paternal allele^,. f | To increase the concentration of a therapeutically relevant gene, Cas proteins can be used to form indels in the microRNA (miRNA) or the 3′ untranslated region, leading to a reduction in miRNA binding and amelioration of the resulting RNA interference^–.