CRISPR Without the Double-Strand Breaks

Since its discovery as a bacterial adaptive immune system, CRISPR has been developed into a powerful gene-editing tool, revolutionizing biological research, and facilitating a greater understanding of many cellular and molecular processes. Conventional CRISPR techniques rely on the generation of site-specific DNA double-strand breaks (DSB) to facilitate gene editing, where a single guide RNA (sgRNA) will guide the Cas9 endonuclease to the target site within the genome where it introduces a DSB. This then triggers endogenous cellular DNA repair pathways, such as non-homologous end joining (NHEJ), an error-prone process that introduces an indel and subsequent frameshift mutation at the target site resulting in robust and efficient gene knockout. Or the DSB will activate homologous repair in the presence of an exogenous template resulting in the introduction of mutations or sequence insertions.¹

The precision, flexibility, and efficiency of CRISPR-Cas9 has seen it overtake previous gene-editing technologies, such as ZFNs and TALENs, as the go-to genome engineering tool. But the introduction of a DSB does come with risks—such as potential effects to cell viability and effects of off-target cleavage, which could be catastrophic for clinical applications. This article looks at the expanding CRISPR toolkit, and the development of other, potentially safer methods that elicit targeted changes to the genome without the introduction of a DSB.

Modulation of the genome using CRISPRi/a

CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) were the earliest CRISPR derivatives capable of modulating the genome without relying on the introduction of a DSB.² Instead, these systems use a catalytically inactive version of Cas9 (dead Cas9 or dCas9) fused to transcriptional repressor or activator domains to inhibit (CRISPRi) or activate (CRISPRa) transcription of a target gene rather than full knockout. Modulation of endogenous gene expression is transient and so can therefore better mimic the effect of therapeutic drugs.³ CRISPRi and CRISPRa can also be used together to identify essential genes—this approach has been used in whole genome screening to identify genetic regulators of disease such as Alzheimer’s Disease and subsequently identify therapeutic targets in the genome.⁴

Nucleotide substitutions with CRISPR base editing

First developed in 2016, CRISPR base editors (BE) are engineered enzyme complexes capable of introducing single-nucleotide substitutions in target genes without introducing a DSB. The BE system utilizes an engineered nickase Cas9 (nCas9) that only cleaves a single strand of DNA—the nCas9 is then fused to a deaminase resulting in a chemical conversion of the base at the target site. The ‘nick’ from the nCas9 triggers cellular DNA repair processes that use the chemically converted base as a template resulting in a CRISPR-mediated nucleotide substitution.⁵ There are currently two BE systems available —cytosine base editors (CBEs) are capable of converting a C to T, while adenine base editors (ABEs) chemically convert A to G. While base-editing options are currently limited by the deaminases available, they have shown great potential as therapeutic candidates as the majority of human genetic diseases are caused by a single nucleotide polymorphism. For example, a CRISPR Cas9-ABE complex has been used to correct the single nucleotide polymorphism responsible for sickle cell disease into a non-pathogenic polymorphism in mice.⁶

Prime editing with CRISPR

Prime editing (PE) is an exciting addition to the CRISPR toolkit, providing the means to introduce substitution, insertions, or deletions into the genome without a DSB and with high precision and efficiency. Prime editing uses nCas9 fused to a reverse transcriptase, effectively allowing a new section of DNA to be written at the target site. The nCas9-RT complex is guided by a pegRNA, a single construct that also contains the edits for the target gene. The nCas9 cleaves the non-complimentary strand of DNA allowing for the pegRNA to bind and serve as the template for the reverse transcriptase. The unedited strand of DNA is then excised by endogenous endonucleases, and the nCas9-mediated nick repaired using the edited strand as the template, resulting in insertions or deletions of up to 50–80 base pairs.⁷ The use of twin prime editing (twinPE) systems can also be used to edit larger sequences, with two pegRNAs targeting opposing strands of DNA.⁸ PE systems show great potential as safe CRISPR-mediated gene-therapies—in vivo studies have shown their utility in correcting disease-causing mutations, such as in cancer-associated mutations of the KRAS gene.¹⁰

PASTE for large-scale edits

PE has since been developed further for even larger-scale gene edits—the programmable addition via site-specific targeting elements (PASTE) system uses nCas9 with bacteriophage serine integrases to insert up to 36 kb of DNA at the target site. Prime editors are used to insert integrase landing sites (AttB) in the target DNA, which are then targeted by integrase protein attachment site (AttP) to facilitate large-scale CRISPR-mediated knock-ins anywhere in the genome.⁹ The large genetic insertions achievable without inducing a DSB with the PASTE system make it an attractive option for gene therapy, especially in instances where genetic disease is caused by mutations of large genes previously recalcitrant to traditional CRISPR editing.

Modulation of the epigenome with CRISPR

The epigenome is defined as the endogenous cellular processes that govern gene expression, including DNA methylation (5mC), histone modifications, and non-coding DNA and RNA.¹¹ By fusing dCas9 to enzymes involved in epigenetic processes, such as methyltansferases and histone acetyltransferases, CRISPR can be used to alter the epigenome and so alter gene expression at the target site. While CRISPR-mediated modulation of gene expression requires further exploration, it has the potential to be applied in therapeutics of epigenetic disorders such as fragile X disease or aging-associated disorders.¹¹

Future developments: CRISPR associated Transposons (CASTS)

Exploration of the flexibility and diversity of bacterial CRISPR systems has led to the discovery of CRISPR-associated transposases (CASTS), thought to result from mobile genetic elements co-opting CRISPR-Cas9 cellular machinery. While previous studies had attempted to fuse the Cas9-sgRNA complex to transposons to confer large-scale sequence insertions, they still often relied on the introduction of the DSB.¹² Recent work has led to the development of a CAST system, which enables large-scale integration of DNA into human cells.¹³ Like the PASTE system, CASTs offer the potential for large-scale genetic integrations into the genome without DSB and so offer an alternative genetic therapy for mutations on large genes.

CRISPR: the evolution continues

Since its development as a gene-editing tool, CRISPR-Cas9 has continually evolved and expanded. A concerted shift in recent years toward methods that omit the DSB and the associated, negative off-target effects has led to the development of a plethora of CRISPR-derived genetic editing and modulation tools. The engineering of nuclease deficient Cas9 and its utilization in CRISPRi/a and epigenetics has led to a greater degree of control and understanding of gene expression in biological processes. Additionally, the development of base editing and prime editing now allow the insertion and deletion of ever larger sequences of DNA into genomes and shows promise in gene-therapies. CRISPR-Cas9 has been invaluable in genetic research and its continual evolution and flexibility cements its role as one of the most successful tools in molecular biology.

Key Takeaways

• Traditional CRISPR gene-editing approaches rely on the introduction of a DSB that come with safety concerns that can limit the application of CRISPR as a gene therapy. But the CRISPR system has since evolved to allow modulation of the genome without inducing DSB.
• CRISPRi/a and epigenetic modulation use a nuclease-deficient Cas9 to exert their desired effects on target genes, leading to greater understanding of gene expression/function and therapeutic targets .
• Base editing, prime editing, and its derivatives TWIN-PE and PASTE utilize other enzymes fused to Cas9 allowing single nucleotide repair to large-scale insertions of genetic material with enormous potential in gene therapy.

References

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