DNA deaminase enzymes play key roles in immunity and have recently been harnessed for their biotechnological applications. In base editors (BEs), the combination of DNA deaminase mutator activity with CRISPR–Cas localization confers the powerful ability to directly convert one target DNA base into another. While efforts have been made to improve targeting efficiency and precision, all BEs so far use a constitutively active DNA deaminase. The absence of regulatory control over promiscuous deaminase activity remains a major limitation to accessing the widespread potential of BEs. Here, we reveal sites that permit splitting of DNA cytosine deaminases into two inactive fragments, whose reapproximation reconstitutes activity. These findings allow for the development of split-engineered BEs (seBEs), which newly enable small-molecule control over targeted mutator activity. We show that the seBE strategy facilitates robust regulated editing with BE scaffolds containing diverse deaminases, offering a generalizable solution for temporally controlling precision genome editing.
The development of split-engineered base editors (seBEs) enables small-molecule control over DNA deaminase activity, decreasing off-target effects and offering a generalizable solution for temporal control over precise genome editing.
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April 10, 2022 8:44 PM
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According to research from the Perelman School of Medicine at the University of Pennsylvania, targeted genome mutations can now be introduced by splitting specific mutator enzymes and then triggering them to reconstitute. The researchers discovered a new gene editing technique that offers controls over other existing techniques and has the potential to be used in-vivo. The technique has been patented and the research is published in the latest issue of Nature Chemical Biology. The research team found that DNA deaminases can be split into two inactive pieces, which can then be reconstituted using a small cell-permeable molecule called rapamycin. The new split base editor system can be introduced and remain dormant in a cell until the small molecule is added, at which point the base editing complex can be rapidly "activated" to modify the genome. This technique would have the potential to control the genetic changes that cause cancer development and growth. It could also be used to identify vulnerabilities in cancer cells.