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

Abstract

CRISPR 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 in Drosophila through 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 culturing Drosophila (18-29 degrees 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 in Drosophila compared to other species. The predictable editing outcome, very high efficiency and minimal byproduct formation of this system allows for near homogeneous biallelic gene inactivation in vivo in a ubiquitous or conditional manner. This work significantly improves our ability to create precise loss-of-function alleles in Drosophila and provides key design parameters for developing highly efficient base editing systems in other ectothermic species.

Comparison of TRIBE and STAMP for identifying targets of RNA binding proteins in human andDrosophilacells

“…Another possible explanation for the lack of APOBEC editing in Drosophila S2 cells was proposed by a recent paper posted on bioRxiv ( Doll et al 2022 ). It indicates that APOBEC deaminase activity is poor at temperatures commonly used for S2 cells (18°–24°) but functional at 29°C; to test this possibility, we repeated the Hrp48-Apobec experiment in S2 cells grown at 28°C.…”

Allele Sails: launching traits and fates into wild populations with DNA sequence modifiers

“…While base and prime editors also have significant levels of editing activity in Drosophila , they are not 100%; instead closer to 36% for a prime editor 35 , and >90% for a base editor 23 . These encouraging rates notwithstanding, it is important to note that the editing enzymes used—nuclease, reverse transcriptase, deaminase, and uracil-DNA glycosylase—are often derived from organisms that live in temperature ranges very different from those in which their use is intended, which may result in significantly reduced activity in the target species 23,36,37 . To explore these less-than-ideal scenarios, we model several representative examples in which the editor is introduced at a frequency of 10% into the wildtype population and has no associated fitness costs, for various editor efficiencies (between 15-100%), both with and without maternal carryover.…”

“…CRISPR nucleases such as Cas9 can cleave and create loss of function (LOF) alleles (especially when using multiple gRNAs) in the Drosophila germline or the plant Arabidopsis thaliana at frequencies near 100% 17,[27][28][29][30][31][32][33][34] . While base and prime editors also have significant levels of editing activity in Drosophila, they are not 100%; instead closer to 36% for a prime editor 35 , and >90% for a base editor 23 . These encouraging rates notwithstanding, it is important to note that the editing enzymes used-nuclease, reverse transcriptase, deaminase, and uracil-DNA glycosylase-are often derived from organisms that live in temperature ranges very different from those in which their use is intended, which may result in significantly reduced activity in the target species 23,36,37 .…”

“…The presence of the editor may result in some fitness cost to carriers [18][19][20] . As such, we explored the context in which the editor but not the edits result in fitness costs.…”

Comparison of TRIBE and STAMP for identifying targets of RNA binding proteins in human andDrosophilacells

“…In this manuscript, the authors fused APOBEC1 to Cas9 in an attempt to use APOBEC as a gene editing enzyme in Drosophila . APOBEC deaminase activity was poor at lower temperatures (18°-24°) but functional at 29°C (Doll et al 2022). This may be the explanation for low APOBEC deaminase in Drosophila S2 cells cultured at 23°C.…”