CRISPR Genome Editing Technology Past, Present and Future

Researchers from the University of Zurich published a review article in the journal Cell titled: Past, present, and future of CRISPR genome editing technologies. Genome editing has become a transformative force in life sciences and human medicine, providing unprecedented opportunities to dissect complex biological processes and fundamentally treat genetic diseases. CRISPR-based technologies, with their remarkable efficiency and ease of programmability, are at the forefront of this revolution. In this review, the authors discuss the current state of CRISPR gene editing technologies in research and therapy, highlighting the limitations that restrict them and the technological innovations developed in recent years to address these issues. In addition, the current applications of gene editing in human health and therapy are examined and summarized. Finally, potential developments that may affect gene editing technologies and their applications in the future are outlined.

Genome editing, the precise and targeted modification of an organism's genetic material, is one of the most significant advances in molecular biology. It has far-reaching applications, from revealing fundamental biological processes to advancing medicine, agriculture, and biotechnology. With the first approval of CRISPR-based therapies for human disease treatments expected by the end of 2023, CRISPR genome editing is entering a new era. This review aims to provide a panoramic view of CRISPR genome editing, highlighting its current status, potential future developments, and obstacles that must be overcome to fully realize its promise in human medicine.

The programmability of CRISPR-Cas nucleases enables them to generate site-specific DNA double-strand breaks, allowing them to be rapidly adapted to genome editing technologies. The canonical Cas9 protein (SpCas9) from Streptococcus pyogenes was the first Cas nuclease to be used for genome editing and remains the most widely used gene editor due to its inherent high activity and specificity. Cas12a, a Cas nuclease derived from the V-type CRISPR-Cas system, was discovered a few years after Cas9 and has also been used for genome editing. Unlike Cas9, Cas12a does not require tracerRNA for activation, a feature that has been exploited for multiple editing in vivo.

Figure 1. Molecular principles of CRISPR genome editing. (Pacesa M, et al., 2024)

Since the initial demonstration of CRISPR-based gene editing, the field has seen unprecedented development. The capabilities of the first generation of DNA double-strand break-dependent genome editors based on Cas9 and Cas12a nucleases have been enhanced by continuous innovations that have not only increased the versatility of these tools, but also improved their precision and minimized the consequences of unintended editing. However, concerns about their safety remain, both because of off-target editing activity and the potential genotoxic effects of targeted DNA double-strand breaks. In order to reduce the occurrence of unintended editing, multiple methods have been explored to precisely control CRISPR genome editors.

In addition, concerns about the genotoxicity of DNA double-strand breaks and the low efficiency of homology-directed repair (HDR) have further promoted the development of "second-generation" CRISPR technologies. These technologies mediate genome editing without relying on DNA double-strand break formation and HDR, the most representative of which are base editors (BE) and prime editors (PE).

Currently available CRISPR genome editing technologies mainly include CRISPR-Cas9, CRISPR-Cas12a, base editing, guide editing, transcription regulation, and RNA editing, which provide a more targeted approach to genome editing, with specific technologies particularly suitable for certain types of editing or delivery modes. But while these new additions to the genome editing toolkit have made significant contributions to addressing many of the limitations associated with standardized CRISPR genome editing, they still have some limitations in terms of editing activity, specificity, and delivery.

Figure 2. Current CRISPR editing technologies. (Pacesa M, et al., 2024)

First, CRISPR has transformed genetic research, enabling scientists to model disease-causing mutations in a variety of experimental models, create large-scale genome-wide screening methods, and develop synthetic gene recording devices to study normal development and disease progression. CRISPR systems have also been used to develop molecular diagnostics that make the detection of viral DNA or RNA specific, rapid, and sensitive.

Second, CRISPR technology has also been used to establish strategies to eliminate viral or bacterial human pathogens, the latter through the development of engineered bacteriophages. A specific example of limiting the spread of pathogens is CRISPR-based gene drives, in which specific inhibitory traits (such as female sterility) are introduced to destroy the insect population that carries the pathogen (primarily mosquitoes that spread diseases such as malaria).

Finally, the past decade of CRISPR genome editing has led to the development of a variety of therapeutic approaches for genetic diseases, some of which have moved from preclinical studies based on cell and animal models to human clinical trials. This includes both in vivo and in vitro therapeutic correction strategies. In vivo therapeutic correction approaches involve delivering gene editing components to affected tissues inside the human body. In contrast, in vitro approaches involve collecting cells from the patient, editing them in the laboratory, and then transplanting the edited cells back into the patient. In addition, in vitro CRISPR editing has also enabled the generation of autologous and allogeneic genome-modified cell therapies, primarily for cancer immunotherapy.

Figure 3. Applications of CRISPR genome editors relevant to human health. (Pacesa M, et al., 2024)

As the limitations of current CRISPR technologies have become increasingly apparent over the past decade, new approaches and methodologies have continued to evolve and optimize to address these limitations and increase the efficiency and versatility of CRISPR-based genome editing. These emerging third-generation tools and technologies include the recently discovered class of compact RNA-guided nucleases, which have been used for DNA double-strand break-based editing and can serve as RNA-guided DNA binding platforms for other genome editors such as base editors and prime editors.

In the field of genome editing, the insertion of large stretches of DNA sequences into the host genome, especially in non-dividing cells that lack homology-directed repair (HDR), remains a major unmet need. In this context, the development of CRISPR-guided recombinases and transposons offers a promising and potentially powerful avenue to fill this technological gap. Retrotransposon-based genome editing technologies and novel methods for editing RNA transcripts have also emerged. Finally, the creation of new genome editing tools continues to be coupled with the development of delivery methods, which poses a significant challenge for therapeutic applications. Overall, these advances reflect the dynamics of the rapidly evolving field of genome editing, where each new approach offers complementary advantages to address the various needs and challenges of genetic manipulation.

Reference

Pacesa M, et al. Past, present, and future of CRISPR genome editing technologies. Cell, 2024, 187(5): 1076-1100.