Role of nucleotide identity in effective CRISPR target escape mutations.

Autor:	Künne T; Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands.; Laboratory of Food Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands., Zhu Y; Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands., da Silva F; Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands., Konstantinides N; Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands., McKenzie RE; Kavli Institute of Nanoscience, Department of Bionanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands., Jackson RN; Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT, USA., Brouns SJ; Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands.; Kavli Institute of Nanoscience, Department of Bionanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2018 Nov 02; Vol. 46 (19), pp. 10395-10404.
DOI:	10.1093/nar/gky687
Abstrakt:	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.
Databáze:	MEDLINE
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