Methods for Scarless, Selection-Free Generation of Human Cells and Allele-Specific Functional Analysis of Disease-Associated SNPs and Variants of Uncertain Significance.
Methods for Scarless, Selection-Free Generation of Human Cells and Allele-Specific Functional Analysis of Disease-Associated SNPs and Variants of Uncertain Significance.
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DOI：
10.1038/s41598-017-15407-4
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发表时间：
2017-11-08
影响因子：
4.6
通讯作者：
Segal DJ
中科院分区：
文献类型：
--
作者：
Coggins NB;Stultz J;O'Geen H;Carvajal-Carmona LG;Segal DJ
随着全基因组关联研究中不断出现的风险位点和从患者测序中确定的意义不确定的变异，需要更好的方法将这些人类遗传发现转化为公共卫生的改善。在这里，我们将CRISPR/Cas9基因编辑与利用KASP（竞争性等位基因特异性PCR）基因分型技术的创新高通量基因分型管道相结合，在约1个月内创建无疤痕的癌症变异等基因细胞模型。我们成功地在HEK239细胞中模拟了我们实验室先前在PALB2基因中发现的两种新变体，从而产生了代表这两种变体的所有三种基因型的等基因细胞。我们还模拟了HCT-116细胞中已知的结直肠癌功能风险SNP rs6983267。使用这种方法，基因编辑水平极低的细胞仍然可以被识别和分离。我们还介绍了一种新的分子检测方法，ChIPnQASO（染色质免疫沉淀和定量等位基因特异性占据），它使用相同的技术在dna -蛋白质相互作用水平上揭示这些变异的等位基因特异性功能。我们证明转录因子TCF7L2与rs6983267风险等位基因的结合优于非风险等位基因。我们的产品线为功能变异的发现和验证提供了一个平台，可以广泛应用于精准医学的发展。
With the continued emergence of risk loci from Genome-Wide Association studies and variants of uncertain significance identified from patient sequencing, better methods are required to translate these human genetic findings into improvements in public health. Here we combine CRISPR/Cas9 gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) genotyping technology to create scarless isogenic cell models of cancer variants in ~1 month. We successfully modeled two novel variants previously identified by our lab in the PALB2 gene in HEK239 cells, resulting in isogenic cells representing all three genotypes for both variants. We also modeled a known functional risk SNP of colorectal cancer, rs6983267, in HCT-116 cells. Cells with extremely low levels of gene editing could still be identified and isolated using this approach. We also introduce a novel molecular assay, ChIPnQASO (Chromatin Immunoprecipitation and Quantitative Allele-Specific Occupation), which uses the same technology to reveal allele-specific function of these variants at the DNA-protein interaction level. We demonstrated preferential binding of the transcription factor TCF7L2 to the rs6983267 risk allele over the non-risk. Our pipeline provides a platform for functional variant discovery and validation that is accessible and broadly applicable for the progression of efforts towards precision medicine.
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影响因子：
12.3
作者：
Cheon JY;Mozersky J;Cook-Deegan R
通讯作者：
Cook-Deegan R
影响因子：
16.6
作者：
Ousterout, David G.;Kabadi, Ami M.;Thakore, Pratiksha I.;Majoros, William H.;Reddy, Timothy E.;Gersbach, Charles A.
通讯作者：
Gersbach, Charles A.
影响因子：
--
作者：
Jin HJ;Jung S;DebRoy AR;Davuluri RV
通讯作者：
Davuluri RV
影响因子：
30.8
作者：
Pomerantz, Mark M.;Ahmadiyeh, Nasim;Jia, Li;Herman, Paula;Verzi, Michael P.;Doddapaneni, Harshavardhan;Beckwith, Christine A.;Chan, Jennifer A.;Hills, Adam;Davis, Matt;Yao, Keluo;Kehoe, Sarah M.;Lenz, Heinz-Josef;Haiman, Christopher A.;Yan, Chunli;Henderson, Brian E.;Frenkel, Baruch;Barretina, Jordi;Bass, Adam;Tabernero, Josep;Baselga, Jose;Regan, Meredith M.;Manak, J. Robert;Shivdasani, Ramesh;Coetzee, Gerhard A.;Freedman, Matthew L.
通讯作者：
Freedman, Matthew L.
DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM