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Role Of Nucleotide Identity In Effective Crispr Target Escape Mutations, Tim Künne, Yifan Zhu, Fausia Da Silva, Nico Konstantinides, Rebecca E. Mckenzie, Ryan N. Jackson, Stan J.J. Brouns

Chemistry and Biochemistry Faculty Publications

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 …