Introduction

The generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro 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 via CRISPR techniques.

Methods

Using a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via 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.

Results

10 rounds of cell-SELEX were conducted via 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.

Conclusions

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.

中文翻译：

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CRISPR 介导的等基因 Cell-SELEX 方法用于生成针对天然膜蛋白的高度特异性适体

介绍

以高特异性结合天然膜蛋白的亲和试剂的产生仍然具有挑战性。大多数体外选择范例利用不同的细胞类型进行阳性和阴性轮次选择（其中阳性选择针对表达所需膜蛋白的细胞，而阴性选择针对缺乏蛋白质的细胞）。然而，这种策略可以产生在靶细胞上结合非预期膜蛋白的亲和试剂。为了解决这个问题，我们通过指数富集 (SELEX) 方案开发了配体的系统进化，该方案利用通过CRISPR 技术生成的等基因细胞对。

方法

使用具有组成型 Cas9 表达的 Caco-2 上皮细胞系，我们通过合成 gRNA 的脂转染敲除SLC2A1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-null 细胞进行 Cell-SELEX 轮次。下一代测序 (NGS) 用于量化预期结合物对野生型细胞的富集。

结果

通过在连续灌注下将 ssDNA 池同时暴露于野生型和 GLUT1-null Caco-2 细胞进行 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剂对 GLUT1 受体的特异性。

结论

我们的数据表明，高度特异性的适体可以通过利用同基因细胞系的 SELEX 策略进行分离。这种方法可广泛用于生成亲和试剂，这些亲和试剂可选择性地结合细胞表面天然构象的膜蛋白。