Advancing Targeted Genome Editing with Base and Prime Editing Technologies

By Deliana InfanteReviewed by Lily Ramsey, LLM

Precision medicine has entered a new age marked by advancements in genome editing, with base editing technology and prime editing technology leading the charge as promising alternatives to the standard CRISPR-Cas9. These innovative approaches are reshaping gene therapy innovations, offering unparalleled accuracy in targeted genetic modifications to combat various diseases.

​​​​​​Image Credit: Natali _ Mis/Shutterstock.com

Introduction

Genome editing has been a transformative force in genetic research and therapy, with the development of CRISPR-Cas9 revolutionizing the ability to make precise changes to DNA.¹ However, the field continues to evolve, with base editing (BE) and prime editing (PE) emerging as advanced forms of genome editing that offer even greater precision.¹

BE and PE technologies were developed by David R. Liu´s group at the Broad Institute of Harvard and MIT^1-2; today, Liu is the founder of Beam Therapeutics³ and Prime Medicine⁴. The first technique developed was BE, which is a form of genome editing that allows for the direct, irreversible conversion of one DNA base into another without the need for double-strand breaks or donor DNA templates.¹

This technology can efficiently target and correct point mutations, which are estimated to account for approximately 30% of known disease-causing mutations.¹ Base editors are engineered to be highly precise, minimizing the likelihood of off-target effects and undesired indels (insertions or deletions) at the target site.¹

On the other hand, Prime editing represents a significant leap forward since it allows for the introduction of virtually any small-scale genetic change, including all possible base-to-base conversions, small insertions, and deletions.¹

The standard CRISPR-Cas9 system relies on double-strand breaks and error-prone DNA repair machinery. PE uses a reverse transcriptase fused to a nickase version of Cas9 and a prime editing guide RNA to direct precise edits. These methods have the potential to correct over 90% of known disease-causing mutations.

For More on Genome Editing

Foundations of Base and Prime Editing

BE and PE are refined versions of CRISPR-Cas9 systems.¹ BE allows for the direct conversion of one DNA base pair into another, effectively changing the genetic code without introducing double-stranded breaks.¹

The key components of base editing include a guide RNA that directs the Cas protein to the target site and a base deaminase enzyme that chemically alters the target base, resulting in a base pair conversion.¹

This method has shown higher editing efficiency and fewer unwanted byproducts, such as insertions or deletions (indels), compared to some other editing methods.¹

Meanwhile, PE expands these capabilities by enabling the insertion or deletion of longer DNA sequences and the repair of harmful mutations without the need for DSBs or donor DNA templates.¹

PE involves a prime editing guide RNA (pegRNA) that guides the Cas protein to the right place in the genome and provides the template for new DNA synthesis.¹ The Cas protein in prime editing is fused to a reverse transcriptase enzyme, which uses the pegRNA template to write the desired edits directly into the target DNA strand.¹

Technological Advancements in Genome Editing

Recent years have seen several breakthroughs in gene editing technologies. One notable advancement is the discovery of the IS200/605 transposon family encoded RNA-guided nucleases.⁵ These proteins introduce a novel mechanism for genome editing, expanding the toolkit beyond traditional Cas9 and Cas12 systems.⁵

Additionally, developments in Cas protein engineering for Protospacer Adjacent Motif (PAM) recognition have broadened CRISPR applications.⁵ Researchers can target a wider range of genomic sequences by modifying Cas proteins to recognize different PAM sequences.⁵

Another significant advancement has addressed the limitations of editing multiple genes simultaneously.⁶ Traditional BE and PE technologies struggled with efficiently editing multiple genomic loci at once. The DAP CRISPR array architecture overcomes this by enabling multiplex base-editing (MBE) and multiplex prime-editing (MPE) in human cells.⁶

This is crucial because many polygenic diseases require simultaneous editing at various genomic sites.⁶ The DAP CRISPR arrays use tRNA as an RNA polymerase III promoter to drive the expression of tandemly assembled tRNA-guide RNA (gRNA) arrays. Then, the gRNAs are released by the endogenous cellular tRNA processing machinery.⁶

This innovative approach has demonstrated the ability to achieve up to 31-loci MBE and up to 3-loci MPE, showcasing the potential of the DAP array in complex genomic applications.⁶

Applications of Base and Prime Editing

The advent of BE and PE have revolutionized the field of genome editing with their novel mechanisms and applications in both research and medicine.⁷

BE and PE offer greater efficiency and reduced genotoxicity compared to traditional CRISPR-Cas9, making them suitable for therapeutic use.⁷ For example, BE has been used in sickle disease research.⁷

Sickle cell disease is caused by a mutation in the β-globin gene (HBB).⁸ BE was applied as a potential treatment for this disease in mice models. A custom adenine base editor was employed to convert the pathogenic allele HBB^S into HBB^G, a non-pathogenic variant of the gene.⁸ These experiments pave the way for potential treatments in humans.

BE and PE approaches have also been applied in cystic fibrosis^9-10 and retinal diseases¹¹; however, their applications are not limited to medical research. They have been applied for editing plant genomes, which makes them a crucial tool for agricultural applications.¹²

Challenges and Ethical Considerations

Base and prime editing technologies offer promising avenues for treating human conditions but present challenges such as ensuring specificity to avoid off-target mutations.¹

In addition, there are important ethical concerns about their use in humans, particularly in the germline, since it can affect future generations and potentially create social disparities.¹³

Future Directions in Genome Editing

Integrating base editing (BE) and prime editing (PE) with omics technologies can provide deeper insights into the functional outcomes of edits, revealing not just genomic changes but also their impact on gene expression patterns and more.¹⁴

This integration is crucial for understanding the systemic effects of genome editing and for developing safer and more effective therapeutic strategies, especially in the context of personalized medicine, where genome editing could tailor treatments to individual genetic profiles.¹⁴

The intersection between genome editing and omics technologies will likely lead to breakthroughs in understanding and treating complex human diseases and even in fields like synthetic biology, where BE and PE could be used to model new biological systems.

References and Further Reading 

1. Testa, L. C., & Musunuru, K. (2023). Base Editing and Prime Editing: Potential Therapeutic Options for Rare and Common Diseases. BioDrugs, 37(4), 453–462. https://doi.org/10.1007/s40259-023-00610-9
2. #WhyIScience Q&A: A molecular biologist builds genome-editing tools to treat genetic diseases. (2023). Broad Institute.  [Online] https://www.broadinstitute.org/news/whyiscience-qa-molecular-biologist-builds-genome-editing-tools-treat-genetic-diseases
3. Therapeutics, B. (n.d.).  [Online]  Breaking new ground to advance science with the potential to change lives. Beam Therapeutics. https://beamtx.com/
4. Prime Medicine | Delivering on the promise of Prime Editing. (2022).  Prime Medicine.  [Online]  https://primemedicine.com/
5. Li Z. H, et al. (2023). Recent advances in CRISPR-based genome editing technology and its applications in cardiovascular research. Military Medical Research, 10(1). https://doi.org/10.1186/s40779-023-00447-x
6. Yuan, Q., & Gao, X. (2022). Multiplex base- and prime-editing with drive-and-process CRISPR arrays. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-30514-1
7. Rottinghaus, M. L. (2024). New kids on the block: Base and prime editors. [Online] https://www.asbmb.org/asbmb-today/science/011724/new-kids-on-the-block-base-and-prime-editors#:~:text=New%20kids%20on%20the%20block%3A%20Base%20and%20prime%20editors
8. Newby G. A, et al. (2021). Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature, 595(7866), 295–302. https://doi.org/10.1038/s41586-021-03609-w
9. Bulcaen M, et al. (2024). Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells. Cell Reports Medicine, 101544. https://doi.org/10.1016/j.xcrm.2024.101544
10. Gene Editing for Cystic Fibrosis. (n.d.).  [Online] Cystic Fibrosis Foundation. https://www.cff.org/research-clinical-trials/gene-editing-cystic-fibrosis#:~:text=Gene%20Editing%20for%20Cystic%20Fibrosis%20%7C%20Cystic%20Fibrosis%20Foundation%20Skip%20to%20main%20content%20Utility%20Search%20Local%20Chapter%20espa%C3%B1ol%20Main%20navigation%20Intro%20to%20CF%20Managing%20CF%20Research%20and%20Clinical%20Trials%20Support%20Community%20Blog%20News%20espa%C3%B1ol%20%5Bseparator%5D%20Get%20involved%20Donate%20Search%20Search%20Gene%20Editing%20for%20Cystic%20Fibrosis%20Cystic%20fibrosis%20is%20caused%20by%20mutations%20in%20both%20copies%20of%20the%20cystic%20fibrosis%20transmembrane%20conductance%20regulator%20%28CFTR%29%20gene
11. Jo D. H, et al. (2023). In vivo application of base and prime editing to treat inherited retinal diseases. Progress in Retinal and Eye Research, 94, 101132. https://doi.org/10.1016/j.preteyeres.2022.101132
12. Molla K. A, et al. (2021). Precise plant genome editing using base editors and prime editors. Nature Plants, 7(9), 1166–1187. https://doi.org/10.1038/s41477-021-00991-1
13. Subica, A. M. (2023). CRISPR in Public Health: The Health Equity Implications and Role of Community in Gene-Editing Research and Applications. American Journal of Public Health, 113(8), 874–882. https://doi.org/10.2105/ajph.2023.307315
14. Testa, L. C., & Musunuru, K. (2023). Base Editing and Prime Editing: Potential Therapeutic Options for Rare and Common Diseases. BioDrugs, 37(4), 453–462. https://doi.org/10.1007/s40259-023-00610-9