Creating stable cell lines with base editing technology and the SH800 Cell Sorter

Base editing: a scientific breakthrough

CRISPR genomics tools offer substantial therapeutic potential due to their precision, adaptability, and unparalleled genome-editing accuracy. Base editing is an valuable technique within this landscape, enabling the meticulous alteration of individual DNA bases—adenine, guanine, cytosine, or thymine—within a target DNA sequence without causing disruptive double-strand breaks [1]. In contrast to traditional CRISPR-Cas9 gene editing, which leverages the cell's natural DNA repair mechanisms, base editing directly converts one DNA base into another [2]. Base editing involves a modified Cas9 protein or Cas9 variant, fused with an enzyme capable of precisely changing the target base. A guide RNA directs this molecular machinery to the intended genomic location, where the base editor orchestrates a chemical modification, delivering a highly precise and predictable DNA sequence alteration.

Base editing is particularly valuable for rectifying single-nucleotide mutations linked to genetic diseases, and for introducing specific genetic modifications with minimal unintended consequences, making it an asset in domains like gene therapy and biotechnology. Pioneered by Professor David Liu and his colleagues at the Broad Institute and Harvard University, base editing has evolved into a transformative technology, with the SH800 cell sorter playing a significant role in establishing stable cell lines for their groundbreaking research endeavors [3-8].

The need for creating stable cell lines in gene therapies

The creation and maintenance of stable cell lines are key steps in aiding rigorous and controlled experimentation in base editing research. This foundation empowers scientists to delve into the functional implications of specific genetic alterations, furthering our comprehension of gene function and regulation. The process of establishing a stable cell line with a CRISPR mutation begins with the meticulous design and synthesis of guide RNAs (gRNAs) targeting the gene of interest. These gRNAs, alongside the Cas9 nuclease or other Cas9 protein, are then introduced into the target cells using techniques like transfection or lentiviral transduction. Subsequently, a precise selection and cultivation of successfully edited cells ensues, employing methods such as limiting dilution or single-cell sorting to create clonal cell lines housing distinct genomic edits. The presence of the intended mutation is confirmed through techniques like PCR, Sanger sequencing, or next generation sequencing, followed by the execution of functional assays to validate the desired phenotypic alterations. It is an ongoing process, involving the continual cultivation and validation of the stable cell line over multiple passages to ensure the persistence of the genomic edit's stability.

The SH800 cell sorter emerged as a foundational tool in this intricate process, supporting Professor Liu’s research for generation of stable cell lines with CRISPR-induced mutations [3-8]. Its functional role lies in efficiently isolating individual edited cells from a diverse and heterogeneous cell population. Following CRISPR editing, cells were genetically engineered to express a selection marker, such as a fluorescent protein or an antibiotic resistance gene. The precision and efficiency of the SH800 enabled the identification and segregation of cells bearing the desired genetic modifications based on these markers. Once isolated, the individual cells were cultured to establish clonal cell lines, a step that ensured the genetic uniformity within each line. This process aided the precise selection of edited cells, underpinning the development of stable cell lines marked by consistent CRISPR-induced mutations. Such precision became a useful asset in this genetic research, and still holds significant relevance in downstream therapeutic applications.

Beyond academia and into the clinic

Following the initial groundbreaking publications [3,4], Professor Liu co-founded Beam Therapeutics (Beam Tx), a biotech company specializing in precision genetic medicine using base editing. Beam Tx is at the forefront of applying this technology commercially, and is actively researching potential applications. Most notably, they administered BEAM-201—a base-edited therapy—to a patient on August 5, 2023, marking the first such treatment in the United States. BEAM-201 is in a Phase 1/2 clinical study for relapsed/refractory T-cell acute lymphoblastic leukemia/T-cell lymphoblastic lymphoma. For more information, visit clinicaltrials.gov (NCT05885464).

Role of Cell Sorting

At Sony Biotechnology, we take great pride in recognizing the pivotal role our cell sorters have played in the development of transformative base editing technology. We believe that cell sorters are instrumental in the creation of stable cell lines, an endeavor of significance in the field of gene therapies. The SH800 cell sorter, in particular, enables the precise isolation of individual cells bearing meticulous genomic edits, ensuring the establishment of homogeneous cell populations—an essential cornerstone for consistent therapeutic outcomes. This precision in cell line production holds immense relevance in the context of gene therapy, in which the accurate delivery of therapeutic genes and precise genetic corrections are of paramount importance. With our cell sorters, researchers can craft dependable and reproducible cellular models, assisting the study of diseases and the development of cutting-edge, gene-based treatments. These innovative tools are effectively charting new frontiers for gene therapies, ushering in the potential for more effective and tailored interventions, ultimately forging a path toward enhanced healthcare and the management of genetic disorders previously considered untreatable.

References

1. Porto EM, Komor AC, Slaymaker IM, Yeo GW. Base editing: advances and therapeutic opportunities. Nat Rev Drug Discov. 2020;19:839-859. PubMed
2. Xue C, Greene EC. DNA repair pathway choices in CRISPR-Cas9-mediated genome editing. Trends Genet. 2021;37:639-656. PubMed
3. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533:420-424.* Nature
4. Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A• T to G• C in genomic DNA without DNA cleavage. Nature. 2017;551:464-471.* PubMed
5. Newby GA, Yen JS, Woodard KJ, et al. Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature. 2021;595:295-302.* PubMed
6. Mayuranathan T, Newby GA, Feng R, et al. Potent and uniform fetal hemoglobin induction via base editing. Nat Genet. 2023;55:1210-1220.* PubMed
7. Sreekanth V, Jan M, Zhao KT, et al. A molecular glue approach to control the half-life of CRISPR-based technologies. bioRxiv. 2023. DOI: 10.1101/2023.03.12.531757* PubMed
8. Trasanidou D, Barendse P, Bouzetos E, et al. Efficient genome and base editing in human cells using ThermoCas9. CRISPR J. 2023;6:278-288.* PubMed

*Publications citing the SH800