Adenine and cytosine base editors (ABE, CBE) are designed to generate single base mutations in genes without necessarily generating DNA double-strand breaks and undesired indel mutations. However, the activity of base editors employing an inactive (dead) SpCas9 is generally low, which may be increased only at the expense of generating undesired indels by using a nickase SpCas9. We have increased the efficiency of dead base editors to a level comparable to that of nickase base editors by enriching cells labelled for efficient base editing using Base Editor Activity Reporter (BEAR), a plasmid-based, fluorescent tool. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we have examined the applicability of increased fidelity variants to decrease Cas9-dependent off-target effects that revealed that CBE remains active on off-targets where increased fidelity mutations and/or mismatches decrease the activity of ABE, making the strategy of applying increased fidelity variants more beneficial for ABE than for CBE.
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