Bayspair's technology was originally developed in order to perform single base nucleotide substitution without leaving editing scars which are difficult to avoid when conventional genome editing tools including CRISPR/Cas9 are used. The scars include not only off-target modifications but also unintended nucleotide changes (including synonymous mutation). Furthermore, the scar includes a trace of selection marker such as LoxP sequence. Bayspair's Scarless Editing does not produce any undesired editing scars, only intended sequence modifications. Our first work was performed on human embryonic stem cells, which are known as very difficult to make desired changes of genomic sequence. The result was published in Nature Methods (Ikeda, et al., 2018. Efficient scarless genome editing in human pluripotent stem cells. Nature Methods, 15, 1045-1047).

The genomic sequence contains not only the coding regions of amino acid sequence but also other regulatory information including functional non-protein coding RNAs and binding sites of gene regulating proteins and RNAs. If any scar occurred in these sequences by conventional gene editing, the genes expression profile could be modified, even if the modification does not change amino acid sequence. In case of human, nearly 90% of all phenotype-associated single nucleotide polymorphisms (SNPs) identified by genome-wide association study lied within non-protein coding regions. If any editing scar occurs at an unintended site, it's very difficult to predict how the scar affects the cell function. Therefore, Bayspair's Scarless Editing technology is very attractive to edit the target genome to produce therapeutic cells and drug discovery tools with fully designed intended genetic modification.

All CRISPR-based genome editing tools (e.g. CRISPR/Cas9, CRISPR/Cpf1, Base Editors or Prime editing) determine the editing position using "guide RNA". Therefore, the guide RNA has to be designed in a way that the genome editing system can recognize an unique target sequence. Otherwise multiple sites including non-target sites of the genomic sequence will be edited. However, it is not always possible to find an appropriate site for the guide RNA target around or on the sequence to be edited. Bayspair's Scarless Editing technology solved this difficulty because it enables "off-guide editing", modification of the sequence apart from the guide RNA target. The conventional editing method using CRISPR/Cas9 is good to disrupt a gene or insert a large size DNA sequence at an approximate position. On the other hand, Bayspair’s Scarless Editing technology is highly adaptable for editing the exact, predetermined site where no good sequence for the guide RNA could be find.

Off-guide editing, one of a superior feature of Bayspair's genome Scarless Editing editing technology, is to deliver maximum performance in plant genome editing. This is because plant genome typically contain a lot of redundant sequences throughout the genome derived from ancient genome duplication during the evolution. Good guide RNA targets are rarely found at or around the sequence to be edited in plant genome.

This type of genomic difference is often seen among varieties within a species, known as single nucleotide polymorphisms (SNPs), and SNPs may determines trait characteristics. In order to edit genomic sequence as depicted above using conventional CRISPR/Cas9 or related technologies such as Base editor or Prime editing, guide RNAs have to be designed for every nucleotide modifications. Bayspair's technology can edit the genomme at multiple sites, like a shotgun targeting, and the modification is fully controlled unlike real shotgun.

DNA marker-assisted breeding, which is still a major method for various trait improvements, is intrinsically transferring of a group of SNPs existing in several kilobases from a donor plant to elite cultivars by crossing the plants, just like the image shown above. Furthermore, DNA marker-assisted breeding typically takes several years with laborious process. It is possible that Bayspair’s Scarless Editing technology simplifies or even eliminates the laborious, time-consuming breeding process.