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Precise and programmable C:G to G:C base editing in genomic DNA
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2020.07.21.213827v1?rss=1
Authors: Chen, L., Park, J. E., Paa, P., Rajakumar, P. D., Chew, Y. T., Manivannan, S. N., Chew, W. L.
Abstract:
Many genetic diseases are caused by single-nucleotide polymorphisms (SNPs). Base editors can correct SNPs at single-nucleotide resolution, but until recently, only allowed for C:G to T:A and A:T to G:C transition edits, addressing four out of twelve possible DNA base substitutions. Here we developed a novel class of C:G to G:C Base Editors (CGBEs) to create single-base genomic transversions in human cells. Our CGBEs consist of a nickase CRISPR-Cas9 (nCas9) fused to a cytosine deaminase and base excision repair (BER) proteins. Characterization of >30 CGBE candidates and 27 guide RNAs (gRNAs) revealed that CGBEs predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean C:G to G:C edits at 15% and up to 37%). CGBEs target cytosine in WCW, ACC or GCT sequence contexts and within a precise two-nucleotide window of the target protospacer. We further targeted genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of CGBE in interrogating and correcting human genetic diseases.
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Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2020.07.21.213827v1?rss=1
Authors: Chen, L., Park, J. E., Paa, P., Rajakumar, P. D., Chew, Y. T., Manivannan, S. N., Chew, W. L.
Abstract:
Many genetic diseases are caused by single-nucleotide polymorphisms (SNPs). Base editors can correct SNPs at single-nucleotide resolution, but until recently, only allowed for C:G to T:A and A:T to G:C transition edits, addressing four out of twelve possible DNA base substitutions. Here we developed a novel class of C:G to G:C Base Editors (CGBEs) to create single-base genomic transversions in human cells. Our CGBEs consist of a nickase CRISPR-Cas9 (nCas9) fused to a cytosine deaminase and base excision repair (BER) proteins. Characterization of >30 CGBE candidates and 27 guide RNAs (gRNAs) revealed that CGBEs predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean C:G to G:C edits at 15% and up to 37%). CGBEs target cytosine in WCW, ACC or GCT sequence contexts and within a precise two-nucleotide window of the target protospacer. We further targeted genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of CGBE in interrogating and correcting human genetic diseases.
Copy rights belong to original authors. Visit the link for more info