A full set toolkit for base editing, prime editing, TR-HDR, and targeted gene insertion
Precise genome editing has become a more demanding issue 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 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 specifies 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 nonhomologous end joining (NHEJ) :
1. Targeted insertion strategy could insert sequences of up to 2,049 base pairs (bp), into the rice genome at an efficiency of 25% .
2. TR-HDR 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 , high efficient targeted insertions, deletion could been achieved through TR-HDR.
Here we developed a web-based software package, Open Genome. A user-friendly, full set toolkit for base editing, prime editing, TR-HDR, and targeted insertion experimental designs based on the principles of the former experiment and provides detailed vector construction methods, time and energy-saving mutation detection methods for CRISPR/Cas9/NG-Cas9/SPRY-based genome editing in both plants and other organisms.
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