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Chimeric DNA:RNA Guide for High Accuracy Cas9 Genome Editing
Chimeric DNA:RNA Guide for High Accuracy Cas9 Genome Editing
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机译:嵌合DNA:高精度Cas9基因组编辑的RNA指南
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摘要
A chimeric DNA:RNA guide for very high accuracy Cas9 genome editing employs nucleotide-type substitutions in nucleic acid-guided endonucleases for enhanced specificity. The CRISPR-Cas9 gene editing system is manipulated to generate chimeric DNA:RNA guide strands to minimize the off-target cleavage events of the S. pyogenes Cas9 endonuclease. A DNA:RNA chimeric guide strand is sufficient to guide Cas9 to a specified target sequence for indel formation and minimize off-target cleavage events due to the specificity conferred by DNA-DNA interactions. Use of chimeric mismatch-evading lowered-thermostability guides (“melt-guides”) demonstrate that nucleotide-type substitutions in the spacer can reduce cleavage of sequences mismatched by as few as a single base pair. The chimeric mismatch-evading lowered-thermostability guides replace most gRNA spacer positions with DNA bases to suppress mismatched targets under Cas9's catalytic threshold.
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