Abstract
Adenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.
Highlights
Adenine and cytosine base editors (ABE, CBE) allow for precision genome engineering
We aimed to develop an easyto-perform and quick gain-of-signal fluorescent assay to monitor base editing activity with a plasmid-based format, that allows the use of numerous sequences and can be adapted to various cell types
Investigating nABE, we found that it tolerates single guide RNAs (sgRNAs) containing one or two mismatches in all the positions examined, with an average of 71% and 37% normalised activity (% of on-target activity), respectively (Supplementary Fig. 6a). dABE exhibited slightly higher fidelity than nABE, which is more apparent with the sgRNAs containing two mismatching positions
Summary
Adenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Cas[9] nucleases recognise DNA sequences that are located immediately upstream of their respective protospacer adjacent motif (PAM) sequences and are complementary to the ~20 nucleotide-long 5’ part (spacer) of their single guide RNAs (sgRNAs)[1,2,3,4] These nucleases facilitate effective genome modifications by introducing site specific double-strand breaks into the DNA; unwanted insertions or deletions (indels) frequently fleck the modified genome[5,6]. Base editors have been designed to perform genome modifications without introducing DNA double-strand breaks They generally comprise an RNA guided nuclease (Cas[9] or Cas12a) fused to a deaminase enzyme, and together they are capable of inducing transition mutations. A detailed description of these developed variants and their features can be found in reference[27]
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