Tang Pei-Zhong, Ding Bo, Peng Lansha, Mozhayskiy Vadim, Potter Jason, Chesnut Jonathan D
Thermo Fisher Scientific Inc., R&D Synthetic Biology, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
Biotechniques. 2018 Nov;65(5):259-267. doi: 10.2144/btn-2018-0105. Epub 2018 Aug 17.
GUIDE-seq was developed to detect CRISPR/Cas9 off-target. However, as originally reported, it was associated with a high level of nonspecific amplification. In an attempt to improve it, we developed target-enriched GUIDE-seq (TEG-seq). The sensitivity level reached 0.1-10 reads-per-million depending on the NGS platform used, which was equivalent to 0.0002-1% measured by Targeted Amplicon-seq. Application of TEG-seq was demonstrated for the evaluation of various Cas9/gRNA configurations, which suggests delivery of Cas9/gRNA ribonucleoprotein results in significantly fewer off-targets than Cas9/gRNA plasmid. TEG-seq was also applied to 22 gRNAs with relatively high in silico ranking score that targeted the biological relevant SNPs. The result indicated the initial selection of gRNAs with high score is important, although it cannot exclude the possibility of off-target.
GUIDE-seq是为检测CRISPR/Cas9脱靶效应而开发的。然而，如最初报道的那样，它与高水平的非特异性扩增有关。为了改进它，我们开发了靶向富集GUIDE-seq（TEG-seq）。根据所使用的二代测序平台，灵敏度水平达到每百万0.1 - 10条 reads，这相当于靶向扩增子测序测量的0.0002 - 1%。TEG-seq的应用被证明可用于评估各种Cas9/gRNA组合，这表明与Cas9/gRNA质粒相比，Cas9/gRNA核糖核蛋白的递送导致的脱靶效应显著减少。TEG-seq还应用于针对生物学相关单核苷酸多态性的22个在计算机模拟中排名分数相对较高的gRNA。结果表明，高分gRNA的初步选择很重要，尽管它不能排除脱靶的可能性。
