Nucleic Acids Research
Oxford University Press
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Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly influence the efficiency of primed CRISPR adaptation. Here we show that guanine-substitutions in the target strand of the protospacer are highly detrimental to CRISPR interference and interference-dependent priming, while cytosine-substitutions are more readily tolerated. Furthermore, we show that this effect is based on strongly decreased binding affinity of the effector complex Cascade for guanine-mismatched targets, while cytosine-mismatched targets only minimally affect target DNA binding. Structural modeling of Cascade-bound targets with mismatches shows that steric clashes of mismatched guanines lead to unfavorable conformations of the RNA-DNA duplex. This effect has strong implications for the natural selection of target site mutations that lead to effective escape from type I CRISPR–Cas systems.
Tim Künne, Yifan Zhu, Fausia da Silva, Nico Konstantinides, Rebecca E McKenzie, Ryan N Jackson, Stan JJ Brouns; Role of nucleotide identity in effective CRISPR target escape mutations, Nucleic Acids Research, Volume 46, Issue 19, 2 November 2018, Pages 10395–10404, https://doi.org/10.1093/nar/gky687