Removal of TREX1 activity enhances CRISPR–Cas9-mediated homologous recombination

Understanding CRISPR–Cas9 DNA Repair Mechanisms

CRISPR–Cas9 induces double-strand breaks (DSBs) at specific genomic loci, triggering the cell’s DNA repair machinery. The two primary pathways for repairing these breaks are non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ rapidly rejoins DNA ends but often introduces insertions or deletions, leading to gene disruption.

In contrast, HDR uses a homologous DNA template to accurately repair the break, allowing for precise gene editing, such as the insertion of therapeutic sequences or correction of mutations. However, HDR is less efficient and is tightly regulated by the cell cycle, predominantly active during the S and G2 phases when sister chromatids are available.

The low efficiency of HDR compared to NHEJ in most human cells presents a major challenge for precise genome editing. Factors such as cell type, cell cycle status, and DNA repair gene functionality influence HDR outcomes, necessitating strategies to enhance HDR for clinical applications.

TREX1: A Key Nuclease Limiting HDR Efficiency

TREX1 is a 3′ to 5′ exonuclease localized to the endoplasmic reticulum and is involved in degrading cytosolic DNA to prevent autoimmune responses. Although primarily studied in innate immunity, TREX1 has recently been identified as a critical factor restricting CRISPR–Cas9-mediated HDR.

Genome-wide CRISPR interference (CRISPRi) screens in lymphoblastic cell lines derived from Fanconi anemia (FA) patients uncovered TREX1 as the sole gene whose knockdown robustly rescues HDR efficiency. This finding implicates TREX1 as a dominant nuclease that degrades exogenous DNA repair templates, thereby limiting the availability of donor DNA for HDR.

The role of TREX1 in reducing HDR is independent of its immune functions. Its nuclease activity targets single-stranded oligodeoxynucleotide (ssODN) templates used for HDR, reducing their cellular stability and consequently lowering precise genome editing efficiency.

Impact of TREX1 Removal on Fanconi Anemia Cells

Fanconi anemia (FA) is a genetic disorder characterized by defective DNA repair pathways, particularly those involved in resolving interstrand crosslinks, leading to bone marrow failure and cancer predisposition. FA patient-derived cells exhibit severely compromised HDR efficiency, limiting CRISPR–Cas9 therapeutic correction.

In studies using FA lymphoblastic cell lines lacking functional FANCD2 or FANCE genes, HDR efficiency was drastically reduced compared to wild-type cells. However, knocking out TREX1 in these FA cells significantly restored HDR levels, highlighting its restrictive role in these deficient DNA repair contexts.

This restoration suggests that targeting TREX1 could overcome intrinsic repair deficiencies in FA and potentially other disorders marked by impaired HDR, enabling more effective genome editing strategies tailored to patient-specific cellular environments.

Mechanisms by Which TREX1 Restricts HDR

TREX1 exerts its effect by degrading exogenous DNA donor templates introduced into cells for HDR. These templates, often single-stranded oligodeoxynucleotides (ssODNs), are susceptible to nucleolytic degradation, which diminishes their availability for homologous recombination.

Moreover, TREX1's localization to the endoplasmic reticulum and its nuclease activity contribute to the rapid clearance of these DNA templates from the cytoplasm before they can efficiently participate in HDR. This limits the formation of the intermediate DNA structures necessary for precise repair.

By removing TREX1 activity, the stability and persistence of HDR templates increase, enhancing the likelihood that the cell’s repair machinery utilizes them for accurate genome editing. This mechanistic insight provides a rationale for improving HDR efficiency through modulation of cellular nucleases.

Chemical Strategies to Protect HDR Templates from TREX1 Activity

Beyond genetic knockout of TREX1, chemical modifications of donor DNA templates offer a practical approach to evade TREX1-mediated degradation. Modifying ssODNs with protective chemical groups can shield them from exonucleolytic activity without compromising their ability to participate in HDR.

Such chemical protection includes modifications at the 5′ and 3′ ends or incorporation of backbone analogs that resist nuclease digestion. These strategies have been demonstrated to rescue HDR efficiency in TREX1-expressing cells, broadening the applicability of genome editing across various cell types.

The combination of chemical template protection and TREX1 modulation represents a promising avenue to enhance precise genome editing, particularly in primary cells or patient-derived models where genetic knockout of TREX1 is not feasible.

Cellular Context and HDR Variability in Genome Editing

HDR efficiency varies widely among cell types and even among cells of the same lineage, influenced by factors such as cell cycle phase, DNA repair gene status, and nuclease expression levels. TREX1 expression contributes significantly to this variability by modulating the availability of donor DNA templates.

For example, identical CRISPR–Cas9 reagents targeting the same genomic locus can yield editing efficiencies ranging from negligible to over 30% depending on the cellular background. Understanding and manipulating factors like TREX1 help explain these stochastic outcomes.

Addressing cellular heterogeneity and nuclease activity is critical for developing universal genome editing protocols with predictable, high-efficiency HDR, facilitating translation from bench to bedside.

Implications for Therapeutic Genome Editing

Enhancing HDR via TREX1 removal or inhibition holds significant promise for therapeutic genome editing, especially for genetic diseases requiring precise correction, such as Fanconi anemia, cystic fibrosis, and muscular dystrophies.

By improving HDR efficiency, gene therapies can achieve higher correction rates with fewer off-target effects and reduced reliance on error-prone NHEJ pathways. This increases safety and efficacy in clinical applications.

Future research may focus on developing small molecule inhibitors of TREX1 or optimizing chemically protected donor templates to create clinically viable strategies that augment HDR without permanent genetic alterations to patient cells.

Conclusion

The discovery of TREX1 as a critical regulator restricting CRISPR–Cas9-mediated homologous recombination marks a significant advancement in genome editing technology. By removing or inhibiting TREX1, researchers can markedly enhance HDR efficiency, overcoming a major barrier to precise genetic modification. This has profound implications for treating genetic disorders characterized by defective DNA repair, such as Fanconi anemia, and for improving the consistency and safety of CRISPR-based therapies. Moving forward, integrating TREX1 modulation with chemical protection strategies for donor DNA templates promises to unlock the full potential of precise genome editing in diverse cellular contexts, accelerating the translation of CRISPR technologies into clinical practice.