Next-generation twin prime editor with La domain fusion enables large genetic insertion and simultaneous treatment of genetic diseases
Background
Gene editing technology has made remarkable progress. In particular, prime editing (PE) technology, which has evolved from the third-generation CRISPR gene scissors, is gaining attention as a next-generation tool that can substitute, insert, or delete desired bases without completely cutting the DNA double strand. Prime editing, which uses a CRISPR module fused with reverse transcriptase (RT), is optimized for precisely editing target base sequences. Furthermore, twin prime editing (twin-PE) technology, which simultaneously provides two complementary reverse transcription templates to induce large-scale gene insertion or deletion, is emerging.
However, existing twin prime editing systems have the disadvantage of low editing efficiency. There are also technical limitations, such as the induction of unintended insertion/deletion mutations (indels) and the difficulty in predicting editing results in advance. In order to apply it practically to drug development or large-scale genome engineering, both efficiency and precision need to be improved.
Key Findings
The Korea Research Institute of Bioscience and Biotechnology (KRIBB) researchers redesigned the existing prime editor components based on SpCas9(H840A)-RT to overcome these limitations. First, they designed a 'La-twin-PE' system by fusing the RNA-binding domain (La domain) of the La protein, which plays a key role in increasing RNA stability, to the prime editor. In addition, the structure of the twin prime editing guide RNA (pegRNA) was refined to increase accessibility to the target site.
This designed La-twin-PE recorded 1.75 ± 0.21 times higher editing efficiency at various gene loci in human-derived cell lines compared to existing technologies. Notably, inaccurate editing, such as damage to non-target base sequences or the occurrence of non-specific mutations, did not increase.
A large-scale gene insertion experiment was also successfully performed to evaluate the practical ability of the new gene scissors. The researchers achieved the successful insertion (knock-in) of a green fluorescent protein (GFP) gene of approximately 2.8 kilobases (kb) into the target genome locus. Furthermore, this technology is applied to the treatment of spinocerebellar ataxia type 3 (SCA3), a representative degenerative brain disease. SCA3 patients have an abnormally long repeat of CAG base sequences within the ATXN3 gene, which causes the accumulation of toxic polyglutamine (polyQ) protein. La-twin-PE showed the result of cleanly cutting out this abnormal polyQ repeat sequence in patient-derived cells.
Significance and Prospects
This study is evaluated as having broadened the versatility of prime editing. While existing gene scissors have focused on editing at the single base level, La-twin-PE provides a wider range of options, such as directly injecting genes of several kilobases into the target locus or cutting out pathogenic genes.
The academic community seems to be paying attention to the molecular design in which the La domain is fused to increase the half-life of pegRNA in cells and stabilize complex formation. It is expected to be a useful framework for developing other types of CRISPR-modified gene scissors in the future.
However, there are also challenges to be solved before entering the clinical stage. The efficiency of inserting large genes of several kb is still not at a level that can be considered practical treatment, so optimization of the vector system for in vivo delivery is required. In addition, the phenomenon that editing efficiency varies depending on the target gene locus also needs to be investigated. Follow-up studies to verify the long-term safety due to off-target effects in vivo are necessary for it to become a true therapeutic agent.
Recent advances in prime editing technologies using CRISPR modules fused with reverse transcriptase (RT) have enabled efficient and precise reprogramming of target genomic sequences. Twin prime editing using two coordinated prime editor complexes is a promising strategy for inducing extensive genomic modifications via reverse-transcribed complementary templates. However, current twin prime editing systems still require improvements in editing efficiency, accuracy, and intended edit predictability. Here, efficiency and precision of twin prime editing were enhanced via engineering and optimizing conventional SpCas9(H840A)-RT-based prime editor (twin-PE) components. A La-domain-fused prime editor (La-twin-PE) and optimized prime editing guide RNAs (pegRNAs) were developed, achieving a 1.75 ± 0.21-fold increase in gene editing efficiency at multiple genomic loci in human-derived cell lines without increasing unintended indel or inaccurate editing. La-twin-PE facilitated efficient ∼2.8 kb GFP transgene knockin at target loci and eliminated the expanded polyQ tract in
La-twin-PE technology can be directly applied to the development of gene therapies for intractable rare diseases that were virtually impossible to treat with existing drugs. In particular, for trinucleotide repeat disorders, such as spinocerebellar ataxia type 3 or Huntington's disease, which are caused by abnormal repeats of specific base sequences, it is possible to provide a fundamental treatment by cutting out the repeat sequences themselves.
Specifically, a promising approach is to collect the patient's stem cells, normalize the pathogenic genes with La-twin-PE in vitro, and then re-implant them in a cell therapy. In addition, the scenario of delivering the manufactured gene therapy material in vivo using adeno-associated virus (AAV) or lipid nanoparticles (LNP) to the patient's central nervous system also adds to its feasibility. This will be a useful breakthrough for intractable diseases with large gene mutations, such as Duchenne muscular dystrophy.