Digested genome sequencing (Digenome-seq) is a highly sensitive, easy-to-carry-out, cell-free method for experimentally identifying genome-wide off-target sites of programmable nucleases and deaminases (also known as base editors). Genomic DNA is digested in vitro using clustered regularly interspaced short palindromic repeats ribonucleoproteins (RNPs; plus DNA-modifying enzymes to cleave both strands of DNA at sites containing deaminated base products, in the case of base editors) and subjected to whole-genome sequencing (WGS) with a typical sequencing depth of 30×. A web-based program is available to map in vitro cleavage sites corresponding to on- and off-target sites. Chromatin DNA, in parallel with histone-free genomic DNA, can also be used to account for the effects of chromatin structure on off-target nuclease activity. Digenome-seq is more sensitive and comprehensive than cell-based methods for identifying off-target sites. Unlike other cell-free methods, Digenome-seq does not involve enrichment of DNA ends through PCR amplification. The entire process other than WGS, which takes ~1–2 weeks, including purification and preparation of RNPs, digestion of genomic DNA and bioinformatic analysis after WGS, takes about several weeks.

消化基因组测序 (Digenome-seq) 是一种高度灵敏、易于执行的无细胞方法，用于通过实验鉴定可编程核酸酶和脱氨酶（也称为碱基编辑器）的全基因组脱靶位点。使用成簇规则间隔的短回文重复核糖核蛋白（RNP；加上 DNA 修饰酶，在碱基编辑器的情况下，在含有脱氨基碱基产物的位点处切割 DNA 两条链）对基因组 DNA 进行体外消化，并进行全基因组测序。 WGS），典型测序深度为 30×。基于网络的程序可用于绘制与靶点和脱靶位点相对应的体外切割位点。染色质 DNA 与无组蛋白基因组 DNA 并行，也可用于解释染色质结构对脱靶核酸酶活性的影响。 Digenome-seq 比基于细胞的方法更灵敏、更全面地识别脱靶位点。与其他无细胞方法不同，Digenome-seq 不涉及通过 PCR 扩增富集 DNA 末端。除了WGS需要大约1-2周之外，整个过程包括RNP的纯化和制备、基因组DNA的消化以及WGS之后的生物信息分析，大约需要几周的时间。