A temperature-tolerant CRISPR base editor mediates highly efficient and precise gene inactivationin vivo

Abstract

SummaryCRISPR nucleases generate a broad spectrum of mutations that includes undesired editing outcomes which attenuate phenotypes and complicate experimental analysis and interpretation. Here, we develop an optimised cytosine base editing system for gene inactivation inDrosophilathrough predictable C-to-T editing and identify temperature as a crucial parameter for base editing efficiency. We find that activity of an evolved version of the most widely used APOBEC1 deaminase is attenuated within the temperature range commonly used for culturingDrosophila(18-29°C) and many other ectothermic species. In contrast, an evolved CDA1 domain functions with remarkable efficiency within the same temperature range. Furthermore, we show that formation of undesired indel mutations and C-to-G/A edits is exceptionally rare inDrosophilacompared to other species. The predictable editing outcome, very high efficiency and minimal byproduct formation of this system allows for near homogeneous biallelic gene inactivationin vivoin a ubiquitous or conditional manner. This work significantly improves our ability to create precise loss-of-function alleles inDrosophilaand provides key design parameters for developing highly efficient base editing systems in other ectothermic species.