Genome-wide target specificities of CRISPR RNA-guided programmable deaminases
| Autor: | Sangtae Kim, Daesik Kim, Sun Heui Yoon, Kyoungmi Kim, Kayeong Lim, Seuk Min Ryu, Jin-Soo Kim |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
APOBEC-1 Deaminase Recombinant Fusion Proteins CRISPR-Associated Proteins Biomedical Engineering Bioengineering Biology Applied Microbiology and Biotechnology Genome 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Bacterial Proteins CRISPR-Associated Protein 9 Cytidine Deaminase CRISPR Humans Point Mutation Clustered Regularly Interspaced Short Palindromic Repeats Guide RNA Base Pairing Whole genome sequencing Genetics Gene Editing Cas9 Genome Human Endonucleases genomic DNA 030104 developmental biology chemistry Mutagenesis Site-Directed Molecular Medicine RNA Human genome 030217 neurology & neurosurgery DNA Biotechnology |
| Zdroj: | Nature biotechnology. 35(5) |
| ISSN: | 1546-1696 |
| Popis: | Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1-nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 ± 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities. |
| Databáze: | OpenAIRE |
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