1. ChIP-seq
基于ChIP-seq的CRISPR/Cas9脱靶鉴定是利用缺乏DNA切割活性的dCas9仍然可以结合DNA的特性进行的[1, 2]。针对dCas9蛋白进行ChIP-seq（染色质免疫共沉淀测序）可以实现对dCas9在全基因组范围的结合情况进行检测，进而评估潜在的脱靶位点。
2. IDLV capture
基于非同源末端连接（NHEJ）的DNA修复过程中，线性的双链IDLV基因组能够优先整合到双链断裂（DSB）这一特性，IDLV capture[3,4]利用CRISPR/Cas9在细胞中引入DSB并同步转染携带抗生素筛选基因的IDLV进行筛选，可以因整合IDLV而获得抗生素耐受的细胞，通过nrLAM-PCR扩增整合位点附近基因组DNA即可确认脱靶位点。
3. GUIDE-seq
GUIDE-seq[5]同样是利用双链DNA断裂（DSB）位点的非同源末端连接（NHEL）过程中可以连入双链DNA的特性，通过引入一种短双链寡核苷酸（dsODN，含硫代磷酸二酯键修饰，用以稳定双链DNA）来标记CRISPR/Cas9切割的DSB，测序这些标签所在的基因组区域，就能够确定脱靶突变的位置。
4. LAM-HTGTS
LAM-HTGTS[6]，即线性扩增介导的高通量全基因组易位测序，是基于CRISPR/Cas9 等造成的DSB可能导致染色质DNA的易位连接，通过检查这些易位连接位点即可以识别CRISPR/Cas9的潜在脱靶位点。但由于出现易位连接的情况很少，这种方法可能会低估脱靶效率。
5. BLESS/BLISS/End-seq/DSBCapture等
BLESS[7]/BLISS[8]/End-seq[9]/DSBCapture[10] 这些方法都是通过直接在DSB位点连入接头，捕获DSB位点，结合高通量测序技术进行脱靶位点检测。
6. DISCOVER-seq
DISCOVER-seq[11]是一种利用 DNA 修复因子（MRE11）结合染色质免疫共沉淀和高通量测序的方法。CRISPR/Cas9在细胞中造成DSB后，MRE11会结合至DSB处进行修复，通过针对MRE11的ChIP-seq可以捕获到相应的DSB位点，进而鉴定到潜在的脱靶位点。
Reference
1. Kuscu, C., et al.,Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.Nat Biotechnol, 2014.
2. Wu, X., et al.,Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.Nat Biotechnol, 2014.
3. Wang, X., et al.,Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors.NatBiotechnol, 2015.
4. Gabriel, R., C. vonKalle, and M. Schmidt,Mapping the precision of genome editing.Nature biotechnology, 2015.33(2): p. 150-152.
5. Tsai, S.Q., et al.,GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.Nature biotechnology, 2015.33(2): p. 187-197.
6. Frock, R.L., et al.,Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases.Nature biotechnology, 2015.33(2): p. 179-186.
7. Crosetto, N., etal.,Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing.Nature methods,2013.10(4): p. 361-365.
8. Yan, W.X., et al.,BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks.Naturecommunications, 2017.8: p. 15058.
9. Canela, A., et al.,DNA Breaks and End Resection Measured Genome-wide by End Sequencing.Molecular cell, 2016.63(5): p. 898-911.
10. Lensing, S.V., et al.,DSBCapture: in situ capture and sequencing of DNA breaks.Nature methods, 2016.13(10): p. 855-857.
11. Wienert, B., et al.,Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq.Science (New York, N.Y.), 2019.364(6437): p. 286-289.
