Gene or genome editing describes a process by which targeted and deliberate changes to the DNA sequence are made. Recent advances in programmable nucleases, including the CRISPR-Cas9 system, mean that manipulating DNA has never been easier, which could fundamentally alter the way we treat and prevent human disease. The repair of genes that cause inherited disorders, deletion of pathogenic drivers for the treatment of infectious disease, and the introduction of therapeutic or protective mutations are all now possible.

This article reviews how the gene-editing landscape has evolved, and the impact CRISPR-Cas9 is having on applications such as gene therapy.

Harnessing cellular DNA repair pathways for gene editing

The basis of gene editing relies on initiating a double-strand break (DSB) at the chromosomal site of interest to trigger one of two endogenous cellular DNA repair pathways. The introduction of a DSB will initiate either the nonhomologous end joining (NHEJ) pathway, which is error prone and so useful for gene disruption due to the introduction of insertion or deletions (indels), or homologous repair (HR), which allows for the integration of a donor sequence at the target site. With the simple introduction of a DSB at the site of interest, the sequence can be manipulated as desired—either deleting the gene of interest or allowing for targeted repair or replacement of specific sequences.

Initial gene-editing attempts utilized naturally occurring nucleases, termed meganucleases, to introduce a DSB for gene-editing applications. Derived from microbial mobile genetic elements, these endonucleases recognize specific 12–40bp sequences and introduce a DSB with high specificity. Using these molecules for gene editing therefore relies on finding the right meganuclease that targets your chosen site—but there is a limited range to choose from.

Protein power with ZFN and TALENs

Gene editing then looked to protein engineering to create custom tools—maintaining the specificity of the meganuclease but aiming to widen the range of targets. By combining several different zinc finger DNA binding domains that target specific sequences, and tethering them to a FOK1 restriction endonuclease, the zinc finger nuclease (ZFN) was developed as the first programmable nuclease for gene-editing purposes. For many years, ZFNs were the only programmable site-specific nuclease available—until the discovery of a simple DNA binding domain from the plant bacteria Xanthamonas, which brought about the development of the next phase of gene-editing technology.

TALENs (transcription activator-like effector nuclease) are created by assembling several TALE DNA binding domains into the desired combination to target specific sequences. The engineered modular molecules are then fused to a nonspecific endonuclease that introduces the DSB at the target site.

Both ZFN and TALEN technologies have provided researchers with the means to edit genes and make targeted changes to DNA in a highly specific and precise manner. However, protein engineering can be challenging and time consuming—it can take years to design and develop a ZFN molecule to target a particular mutation, which is not cost effective in the face of thousands of disease-causing mutations.

Enter CRISPR-Cas9

In 2013, a new gene-editing tool was developed—one that did not require protein engineering to direct the nuclease to the target site but rather just a short piece of RNA. The CRISPR-Cas9 system (which stands for clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) was originally part of a microbial immune system used to defend against foreign DNA but has since been adapted as a powerful gene-editing tool.

The CRISPR-Cas9 system consists of a short guide RNA (sgRNA) that directs the Cas9 endonuclease to the target site of interest—unlike ZFN and TALENs that require extensive protein engineering, retargeting the Cas9 requires only a change to the 20-nucleotide portion of the sgRNA complementary to the target loci. The simplicity of the system and ease of use has contributed to its rapid adoption across the biological space. The use of a catalytically inactive version of Cas9 has also expanded the gene-editing toolkit. By tethering dCas9 to a range of regulatory regions, CRISPR-Cas9 can be used to elicit other genetic changes such as modulation of gene expression with CRISPRi and CRISPRa, and epigenetic modifications such as base editing.

CRISPR-Cas9: a new tool for gene therapy?

The ability to mediate gene editing in virtually any organism and cell line with CRISPR-Cas9, both in vitro and in vivo, is having a tremendous impact across many areas of biological research and model systems, including development of disease models, identification of cellular pathways, and drug discovery. With recent improvements to the specificity of CRISPR-Cas9 and a reduction in off-target effects, there is also great interest in the potential of CRISPR-Cas9 in gene therapy.

Conventional gene-therapy approaches aim to insert a healthy copy of a gene to replace or offset the effect of the disease-causing version. This typically involves the transfer of the nonmutated gene spliced into a viral vector, which will then randomly integrate into the genome. But there are drawbacks—there is no certainty the exogenous gene will even be expressed and it could even have a deleterious effect by silencing a normal gene, or even switching on an oncogene. Gene editing therefore allows for a cut-and-paste strategy as the defect in the mutated gene can be removed and replaced. Recent trials utilizing CRISPR-Cas9 for gene therapy have focused on cancer and blood disorders trial at the University of Pennsylvania is performing gene editing on T cells to program them to attack cancer cells in patients with recurring myeloma and sarcoma. And work is ongoing in patients with sickle-cell disease and beta-thalassemia, where CRISPR-based gene therapy is being used to replace the defective genes that cause disease.

Gene editing 2.0

The CRISPR-Cas9 system has proven to be so simple to implement and flexible for effective gene editing that it has seen a truly remarkable level of adoption. Since its debut in 2013, the number of publications featuring CRISPR-Cas9 already outnumbers those for ZFN and TALENs combined. But these current technologies rely on the introduction of a DSB to facilitate gene editing—and relying on the cellular DNA repair machinery can make it difficult to control the resulting edits across different cells.

In a recent paper published in Nature, Anzalone et al present an alternative CRISPR-based method called prime editing that does not require a DSB and offers improved precision and editing control. Prime editing involves a modified dCas9 fused to a reverse transcriptase, which is guided to the target site by a prime-editing guide RNA (pegRNA) where it introduces a single-strand break that is then repaired by the reverse transcriptase. The authors report that they have used prime editing to perform more than 175 different types of edit in human cells—including insertions, deletions, and point mutations, all without requiring DSB or donor templates.

The CRISPR revolution

The development of gene-editing technologies, from ZFN and TALENs to the CRISPR-Cas9 system, has ushered in an exciting era in biology. The simple-to-use CRISPR-Cas9 system has provided researchers with a powerful tool with which to perform gene editing in a myriad of different cell lines and organisms. Researchers can now make targeted modifications to DNA sequences relatively easily, allowing the study of gene function and interactions in the cellular context, and opening up possibilities in the way we treat and prevent human disease.

Key Takeaways

  • Gene editing relies on activating cellular DNA repair pathway by introducing a double-strand break to the DNA at the chosen site
  • ZFN and TALENS were the first programmable nucleases to be developed for gene editing – but require protein engineering, which can be challenging
  • The RNA-based CRISPR-Cas9 system is a powerful gene-editing tool allowing precise and targeted manipulations to genetic sequences to be made in virtually any organism and cell line
  • With improvements to specificity and a reduction in off-targets effects, CRISPR-Cas9 is being developed as a possible gene therapy treatment