Optimizing twin prime editing components for scalable genome editing and therapy in spinocerebellar ataxia type 3

초록

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 ATXN3 in an engineered human-derived mutant cell line modeling spinocerebellar ataxia type 3. The optimized twin prime editing platform facilitates highly efficient and scalable genomic engineering through streamlined pegRNA design, offering substantial potential for a broad spectrum of biotechnological and therapeutic applications.

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