Base Editing: Expanding the Gene Editing Toolbox

Fast-paced developments and progress in the field of molecular genetics have greatly expanded our understanding of genes and their function in health and disease. This knowledge reinforces the need for continued innovation and inspires scientists to seek ways to modify DNA sequences directly in a precise and controlled fashion to advance human therapeutics. Such control over the genetic code of an organism holds great promise: from boosting fundamental research in molecular biology and disease modeling to opening new avenues in cell and gene therapy by correcting disease-causing mutations. The discovery of CRISPR-Cas9 revolutionized the field and propelled innovation efforts to a whole new level. Base editing is one of the latest CRISPR-Cas-derived genome editing agents, promising access to safer, easier, and more precise genome editing.

Enter base editing

Base editing is a next-generation, CRISPR-based precision gene-editing technology developed to circumvent the intrinsic limitations of “traditional” CRISPR-Cas9. By fusing or recruiting a deaminase to a Cas9 nickase (nCas9), base editors generate targeted point mutations on DNA via chemical modification (hydrolytic deamination), rather than physical replacement, and subsequent cellular processing of target nucleobases. The R-loop “bubble” generated on the target locus upon binding of the nCas9 and its associated single guide RNA reveals a stretch of single-stranded DNA, which serves as the natural substrate for the recruited deaminase. Two main classes of base editors have been developed: cytosine base editors (CBEs) that convert C–G base pairs into T–A through a uracil intermediate, and adenine base editors (ABEs) that convert A–T into G–C via an inosine intermediate.

Image: CRISPRko editing vs. Base editing

Base editing allows the introduction of single amino acid substitutions, or seamless disruption of target gene expression via the generation of stop codons or disruption of splice sites. As base editors do not introduce double-strand breaks, like “traditional” CRISPR-Cas systems do, the risk of triggering cytotoxic effects or potentially oncogenic alterations in the genome, such as chromosomal translocations or other aberrations, is considerably reduced. This opens the possibility of using base editors efficiently for the introduction of multiple, simultaneous edits via multi-gene targeting without escalating these risks, nor compromising cell viability and functionality. These safety features, along with the fact that single nucleotide variants (SNVs) underlie the pathogenesis of most human genetic diseases, make base editing extremely attractive for cell and gene therapy applications.

Base editing in cell and gene therapy

From Duchenne muscular dystrophy¹ to Hutchinson-Gilford progeria², CBEs and ABEs have been employed in proof-of -concept in vitro or in vivo studies looking at correcting SNVs involved in the pathogenesis of several genetic conditions. In cancer immunotherapy, base editing is being explored for the generation of universal, off-the-shelf allogeneic CAR-T cells, with the promise of making CAR-T treatments safer, faster to manufacture, and less costly³ with some base editors already set to enter the clinic. In November last year, Beam Therapeutics’ Investigational New Drug BEAM-101, an ex vivo autologous hematopoietic cell therapy to treat sickle cell disease, was cleared by the U.S. Food and Drug Administration to enter a Phase I/II clinical trial.⁴ Only five years since base editing was first described, this proves just how fast the technology is sparking interest and breaking boundaries in the therapeutic gene editing space, while also providing new hope for the future treatment of debilitating genetic diseases.

Future developments

Together, CBEs and ABEs can theoretically address and correct 69% of human SNVs associated with monogenic disorders.⁵ However, the actual number of modifiable genetic loci is limited by the protospacer adjacent motif (PAM) requirement of Streptococcus pyogenes Cas9, the nCas9 used in CBEs and ABEs. The use of alternative or engineered Cas enzymes with less stringent or alternative PAM requirements will expand the targeting scope of base editors by widening the accessible loci in the genome.

Intensive research and development efforts are focused on increasing the efficiency and product purity of base editors. Owing to the specific, intrinsic properties of the deaminase enzyme and steric constraints within the R-loop, the activity of each base editor tends to be restricted to a narrow window within the single-stranded DNA protospacer region. However, the presence of more than one cytosine (for CBEs) or adenine (for ABEs) near the target nucleotide means that additional bases are prone to deamination, resulting in “bystander editing”. Depending on the application, this compromise over product purity can be tolerated or detrimental. The use of alternative deaminases, with a reduced activity window, or re-engineering of the entire base editor architecture are ways to exploit different steric hindrances within the system to achieve narrower or specific spatial deamination profiles. The use of high-fidelity Cas variants is one promising approach aimed at decreasing the chances of off-target editing in the genome, adding another layer of safety to the system.

On the horizon

Base editing has proven itself to be a promising addition to the gene-editing toolbox, with immense potential in gene therapy and beyond. As the technology gains wider adoption, the potential for further improvements and applications increases. Given the rate at which innovation in the field of genetic engineering has been accelerating, with base editing moving from R&D to the clinic in under five years, optimism toward the development and successful use of improved CBEs and ABEs is high. An expanded, safer, and more precise genome-editing toolbox holds great promise for the treatment of hugely debilitating genetic diseases, including non-communicable disorders requiring multiplexed editing to modify two or more DNA loci in the genome.

References

1. Chemello F, et al. Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing. Sci Adv. 2021; 7(18). doi: 10.1126/sciadv.abg4910

2. Koblan L, et al. In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice. Nature. 2021; 589, 608-614. doi: 10.1038/s41586-020-03086-7

3. Webber BR, et al. Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors. Nat Commun. 2019, 5222

4. Beam Therapeutics. BEAM-101 IND Cleared by FDA for Evaluation as a Treatment for Sickle Cell Disease. 2021.

5. Lavrov A, et al. Genome scale analysis of pathogenic variants targetable for single base editing. BMC Med Genomics. 2020, 13(8): 80. doi: 10.1186/s12920-020-00735-8

Andrea Frapporti, Ph.D., is a Senior Scientist in the Base Editing Department at Horizon Discovery, a Revvity company.