Rosch Jonah C, Neal Emma H, Balikov Daniel A, Rahim Mohsin, Lippmann Ethan S
Department of Chemical and Biomolecular Engineering, Vanderbilt University, PMB 351604, 2301 Vanderbilt Place, Nashville, TN 37235-1604 USA.
Department of Biomedical Engineering, Vanderbilt University, Nashville, TN USA.
Cell Mol Bioeng. 2020 Sep 11;13(5):559-574. doi: 10.1007/s12195-020-00651-y. eCollection 2020 Oct.
The generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated CRISPR techniques.
Using a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the gene (encoding the GLUT1 glucose transporter) lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells.
10 rounds of cell-SELEX were conducted simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor.
Our data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach may be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.
生成能以高特异性结合天然膜蛋白的亲和试剂仍然具有挑战性。大多数筛选范式在阳性和阴性筛选轮次中使用不同的细胞类型（其中阳性筛选针对表达所需膜蛋白的细胞，阴性筛选针对缺乏该蛋白的细胞）。然而，这种策略可能会产生与靶细胞上非预期膜蛋白结合的亲和试剂。为了解决这个问题，我们开发了一种利用CRISPR技术生成的同基因细胞对的指数富集配体系统进化（SELEX）方案。
我们使用具有组成型Cas9表达的Caco-2上皮细胞系，通过用合成gRNA进行脂质转染来敲除基因（编码GLUT1葡萄糖转运蛋白）。使用单链DNA（ssDNA）文库对野生型和GLUT1缺失细胞进行细胞SELEX轮次。使用下一代测序（NGS）来量化与野生型细胞的预期结合物的富集情况。
在连续灌注下，将ssDNA库同时暴露于野生型和GLUT1缺失的Caco-2细胞，进行了10轮细胞SELEX。通过流式细胞术和免疫染色验证了从NGS中鉴定出的顶级结合物对GLUT1受体的特异性。
我们的数据表明，利用同基因细胞系的SELEX策略可以分离出高度特异性的适体。这种方法可能广泛用于生成能选择性结合细胞表面天然构象膜蛋白的亲和试剂。
