A full set toolkit for base editing, prime editing, TR-HDR, and targeted gene insertion
Precise genome editing has become a more demanding issue for precision plant breeding since the emerging and rapid development of CRISPR methods. Methods such as adenine and cytosine base editors, prime editing, and recently developed TR-HDR all have equipped this field. Base-editing, based on the CRISPR/Cas9 systems, containing two types of base editors-cytosine base editor (CBE) and the adenine base editor (ABE). The cytosine base editor (CBE) converts C•G to T•A, adenine base editor (ABE) empowers the conversion of A•T to G•C, enabling the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template (Komor et al. 2016) (Gaudelli et al. 2017). Prime editing, developed in 2019, have been making huge improvement of precise genome editing. It directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specify the target site and encodes the desired edit, enabling targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. (Anzalone et al. 2019). Targeted insertion and TR-HDR (tandem repeat-HDR strategy), developed in 2020, based on the design of the phosphorothioate-linkage modification to stabilize the oligos in cells and the 5ʹ-phosphorylation to facilitate non-homologous end joining (NHEJ): Targeted insertion strategy could insert sequences of up to 2,049 base pairs (bp), into the rice genome at an efficiency of 25%. TR-HDR is mainly based on the preview formation of tandemly arranged repeat elements (such as the YFFP and GUUS reporters that contain a tandem repeat of the middle part (F and U, respectively) of the YFP and GUS reporters) using modified donor insertion in plant cells. The oligo is designed to also form a target site for the same sgRNA upon insertion. All 12 types of point mutation, highly efficient targeted insertions, deletion could be achieved through TR-HDR.
Figure 1: Four different precise genome editing methods.
The following list provides a quick overview of the steps involved in precise genome editing design (Figure 2). Note: Read the step-by-step guide to ensure optimal results.
Figure 2: The overall workflow of OpenGenome Toolkit.
Total reaction volume:10ul
TR-HDR modified donor forward:
TR-HDR modified donor reverse:
Knock in modified donor forward:
Knock in modified donor reverse:
Then transfer via gene gun.
Figure 3: Vector construction using ABEmax-nCas9; CBEmax-nCas9; pCBSG032; pCXPE3
Figure 4: Primer design using the Open Genome web tool.
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