Bingo™ Prime Editing Platform
EDITGENE developed Bingo™ Prime Editing point mutation platform, which can perform accurate and efficient gene point mutation in cells. Bingo™ platform is based on the most efficient and accurate point mutation technology - Prime Editing (PE). EDITGENE has over a decade of gene editing experience, and we have summarized and optimized the targeting strategy design principle from hundreds of gene-editing projects. The positive rate is far higher than that of the traditional HDR-based point mutation system.
What is Prime editing?
Prime editing (PE) is a more efficient and accurate system for CRISPR point mutation introduction. Unlike traditional CRISPR/Cas9 gene editing technology, Prime editing uses a novel Cas9 enzyme - Cas9 nickase (Cas9n), which only cuts one strand of double-stranded DNA. This way, no random mutation repair exists during DNA repair.
Prime editing (PE) is a search and replace process. Design a pegRNA (prime editing guide RNA) with gRNA and gene editing template sequence. pegRNA contains specific primer sequence (similar to sgRNA) and a prime editing protein. The specific primer sequence can recognize the target sequence. The prime editing protein is the fusion protein of cas9 nickase and Reverse transcriptase (RT). The primer sequence of pegRNA guides Cas9 nicknase to cut single strand DNA, and reverse transcriptase synthesizes new DNA sequence according to unpaired pegRNA sequence, and in situ synthesizes the edited sequence to replace the original sequence.
The PE system consists of prime editing proteins (Cas9 nickase, Reverse transcriptase) and pegRNA. Cas9 nickase is a Cas9 H840A nickase that can cut single strands of DNA; Reverse transcriptase (RT) is M-MLV; The fusion protein can improve gene editing efficiency, known as PE1. By introducing a mutation to RT, the binding ability between template and PBS can be improved, thus improving the activity and stability of the enzyme. This is called PE2. The editing efficiency of PE2 is increased by 2.3-5.1 times.
pegRNA is a guide RNA containing a reverse transcription template that complements the target genome sequence as a Prime binding site (PBS), and the position where the PBS binds to the target sequence is the starting point of reverse transcription.
With Cas9n, Reverse transcriptase and pegRNA, the prime editing technology can achieve four kinds of transition mutation (C → T, G → A, A → G, T → C) and eight kinds of transversion mutation (C → A, C → G, G → C, G → T, A → C, A → T, T → A, T → G, T → G), truly achieving precise and versatile gene editing. In addition to single base pair mutations, prime editing can also introduce up to 44 bp insertion and up to 80 bp deletion.
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1. Design a pegRNA with a transcription template (carrying the desired point mutation) and the necessary Prime Binding Site (PBS).
2.Under the guidance of pegRNA, Cas9 cleaves single-stranded DNA, and the PBS at the 3' terminus of pegRNA can recognize and pair with the complementary sequence before the cutting site.
3. Reverse transcriptase (M-MLV RT) uses the designed template sequence behind the PBS sequence on the pegRNA as the template for reverse transcription, and directly polymerizes the target sequence onto the cut DNA.
4. The edited DNA double-strand extends a DNA Flap, activating the cell's 3' flap - 5' flap acquisition mechanism to remove the original DNA fragment.
5.Unmatched DNA double strands are repaired through a mismatch repair (MMR) mechanism to repair non-edited strands, and the entire editing process is completed.
Related products
● CRISPR point mutation cell line service : Optimized PE system, achieving a positive rate of >85%. More efficient than the traditional CRISPR/Cas9 point mutation. No off-target, more accurate!
● Premade CRISPR Cas9 point mutation cell line : The newly launched premade point mutation cancer models cover the most common cancer-related mutations. Useful for basic cancer research, cancer diagnosis, and treatment.
