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Rapid generation of long, chemically modified pegRNAs for prime editing

Nature Biotechnology. 2024-09; 
Xinlin Lei , Anhui Huang , Didi Chen , Xuebin Wang , Ruijin Ji , Jinlin Wang , Yizhou Zhang , Yuming Zhang , Shuhan Lu , Kun Zhang , Qiubing Chen , Ying Zhang , Hao Yin
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Custom DNA/RNA Oligos To express the MLH1dn protein, we generated an E. coli codon-optimized MLH1dn sequence (GenScript) and cloned it into the pET28a-His expression vector17. Get A Quote
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摘要

The editing efficiencies of prime editing (PE) using ribonucleoprotein (RNP) and RNA delivery are not optimal due to the challenges in solid-phase synthesis of long PE guide RNA (pegRNA) (>125 nt). Here, we develop an efficient, rapid and cost-effective method for generating chemically modified pegRNA (125-145 nt) and engineered pegRNA (epegRNA) (170-190 nt). We use an optimized splint ligation approach and achieve approximately 90% production efficiency for these RNAs, referred to as L-pegRNA and L-epegRNA. L-epegRNA demonstrates enhanced editing efficiencies across various cell lines and human primary cells with improvements of up to more than tenfold when using RNP delivery and several hundredfold with RNA d... More

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