On March 17, 2026, Prof. Wensheng Wei’s team from Peking University and Changping Laboratory published a review article titled "Decoding disease-relevant variants with base and prime editors at scale" in Trends in Biochemical Sciences. This paper systematically outlines the development of precision genome editing tools, focusing on how they drive high-throughput functional screening to decipher disease-associated variants at a large scale at the single-base level, reveal mechanisms of drug resistance and immune evasion, decode basic biological processes, and promote the translation of related discoveries into precision diagnostics and therapeutics.

In the human genome, the functional resolution of a massive number of variants of uncertain significance (VUS) has long been a core challenge in functional genomics. The emergence of base editors (BE) and prime editors (PE) has provided a revolutionary solution to this problem. These tools enable programmable point mutations and small insertions or deletions (indels) in situ in the genome without causing DNA double-strand breaks, thereby empowering high-throughput, high-resolution functional screening.

The precision revolution in functional genomics

In the post-GWAS era, precisely elucidating how individual genetic variants affect cellular functions and disease progression has become a key scientific question. Traditional CRISPR/Cas9 knockout screening relies on random repair following DNA double-strand breaks, which presents limitations such as unpredictable repair outcomes, potential cytotoxicity, and the inability to achieve precise single-base manipulation.

Base editors fuse a catalytically impaired Cas9 with a deaminase to achieve programmable base conversions, mainly including cytosine base editors (CBE) and adenine base editors (ABE) ^1,2. Prime editors further fuse a Cas9 nickase with a reverse transcriptase, and under the precise guidance of a pegRNA, can achieve all 12 types of base substitutions as well as small indels³. These technologies avoid the reliance on donor templates and inefficient homology-directed repair, moving gene editing into a more precise and predictable new stage. With the continuous iteration of these tools (Figure 1), their editing efficiency and application scope have significantly improved, making systematic saturation mutagenesis possible and laying a solid foundation for the large-scale decoding of genetic variants.

Figure 1. Schematic of representative base editors and prime editors

Decoding the functional map of disease-relevant variants

One of the core applications of precision editing platforms is to conduct systematic mutational scanning of disease-relevant genes to construct high-resolution functional maps, thereby establishing a direct link between genetic associations and biological effects (Figure 2).

In cancer research, using BEs/PEs to perform saturation mutagenesis screening on key tumor suppressor genes such as BRCA1, BRCA2, and TP53 has successfully identified a large number of functional variants that affect DNA repair or drive tumorigenesis⁴. Combined with cutting-edge technologies like single-cell transcriptomics, such screens can also resolve mutation-induced transcriptional changes in complex cellular environments⁵. Furthermore, precision editing screening has expanded from single genes to pathways and networks. By constructing large-scale mutation libraries encompassing databases like MSK-IMPACT, ClinVar, and COSMIC, researchers have systematically analyzed key pathways such as DNA damage repair⁶. In addition, this strategy has been applied to mechanistic studies of hematological disorders (e.g., GATA1 mutations) and metabolic diseases (e.g., LDLR mutations), continuously expanding the cognitive boundaries of clinical genetics.

Figure 2. Application of base and prime editor screens in systematically characterizing functional variants

Revealing mechanisms of drug resistance and immune evasion

Beyond decoding pathogenic variants, precision editing screening also shows significant value in uncovering the genetic basis of drug response and tumor immune regulation.

In drug-based tumor therapy, saturation mutation at the amino acid level for key targets such as BCL2, MEN1, EGFR, and KRAS can prospectively construct resistance maps, identifying mutations that weaken drug binding while maintaining protein function, providing an important basis for drug resistance mechanism research, new drug development, and efficacy prediction. In the field of tumor immunotherapy, high-throughput screening is used to systematically mine key mutations affecting antigen presentation and immune checkpoint regulation, revealing intrinsic tumor immune evasion mechanisms. Notably, this strategy has been successfully applied to the engineering of primary T cells; by screening for gain-of-function mutations, the persistence and killing activity of T cells are significantly enhanced, providing a new path for next-generation adoptive cell therapy⁷.

Deciphering basic biological regulatory mechanisms

Precision editing technologies also provide powerful tools for basic biological research.

Using this platform, researchers have systematically resolved the molecular mechanisms of key processes such as DNA replication fidelity and RNA splicing regulation. Furthermore, large-scale screening targeting specific amino acid sites has revealed numerous key sites involved in post-translational modifications of proteins (such as ubiquitination, acetylation, and phosphorylation). This technology has also advanced the in-depth study of epigenetic regulation, such as constructing single-amino-acid functional maps of proteins like DNA methyltransferases, and resolving the mechanistic role of m6A modification in cell fate determination. Meanwhile, precision editing is reshaping our understanding of the "dark genome": single-base scanning of non-coding regulatory elements has identified numerous key regulatory sites; and the systematic evaluation of synonymous mutations indicates that they can produce functional effects by affecting mRNA splicing or stability, thereby correcting the traditional perception that "synonymous mutations are harmless"⁸.

Current challenges and future perspectives

Although precision editing screening has made significant progress, it still faces challenges such as the heterogeneity of editing efficiency, bystander editing within the editing window, and potential off-target effects, which impose higher demands on large-scale data interpretation. By optimizing library design, introducing "sensor" systems, and combining computational models for efficiency correction, data reliability can be improved to a certain extent. Meanwhile, protein engineering and artificial intelligence (AI)-driven design are continuously generating a new generation of highly efficient and specific editing tools.

Looking ahead, the deep integration of precision editing technologies with single-cell multi-omics, structural biology, and AI will further propel the development of functional genomics. By providing systematic and mechanistic annotations of genetic variants, this technological framework is expected to continuously deepen our understanding of human physiological and disease processes, leading biomedical science into a more predictable and programmable new era.

Dr. Ying Liu (co-corresponding author, Associate Researcher at Changping Laboratory) and Dr. Xuran Niu (postdoctoral fellow at Peking University) are the co-first authors of this article. This research was supported by the National Natural Science Foundation of China, the Peking-Tsinghua Center for Life Sciences, Changping Laboratory, and the Beijing Nova Program.

Paper link：https://www.cell.com/trends/biochemical-sciences/abstract/S0968-0004(26)00029-0

References:

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8.       Niu, X. et al. (2025) Prime editor-based high-throughput screening reveals functional synonymous mutations in human cells. Nat. Biotechnol. https://doi.org/10.1038/s41587-025-02710-z