title: Genome-wide target specificities of CRISPR RNA-guided programmable deaminases.
abstract: 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.
authors.affiliation: Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
authors.forename: Daesik
authors.initials: D
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Seoul National University, Seoul, Republic of Korea.|Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.
authors.forename: Kayeong
authors.initials: K
authors.lastname: Lim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.
authors.forename: Sang-Tae
authors.initials: ST
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
authors.forename: Sun-Heui
authors.initials: SH
authors.lastname: Yoon
authors.affiliation: Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.
authors.forename: Kyoungmi
authors.initials: K
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Seoul National University, Seoul, Republic of Korea.|Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.
authors.forename: Seuk-Min
authors.initials: SM
authors.lastname: Ryu
authors.affiliation: Department of Chemistry, Seoul National University, Seoul, Republic of Korea.|Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.|Department of Functional Genomics, University of Science and Technology, Daejeon, Republic of Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
mesh_terms: D000071479:APOBEC-1 Deaminase / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D020029:Base Pairing / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003564:Cytidine Deaminase / Q000235:genetics; D004720:Endonucleases / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D017354:Point Mutation / Q000235:genetics; D012313:RNA / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D011993:Recombinant Fusion Proteins; D012313:RNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D000071479:APOBEC-1 Deaminase; C000605089:APOBEC1 protein, human; D003564:Cytidine Deaminase