CasPlay 提供了一个基于 gRNA 条码的 CRISPR 展示平台，用于抗体库分析。

CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling.

机构信息

Division of Genetics, Brigham and Women's Hospital, Howard Hughes Medical Institute, Boston, MA 02115, USA.

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

出版信息

Cell Rep Methods. 2022 Oct 17;2(10):100318. doi: 10.1016/j.crmeth.2022.100318. eCollection 2022 Oct 24.

DOI:10.1016/j.crmeth.2022.100318

PMID:36313802

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9606310/

Abstract

Protein display technologies link proteins to distinct nucleic acid sequences (barcodes), enabling multiplexed protein assays via DNA sequencing. Here, we develop Cas9 display (CasPlay) to interrogate customized peptide libraries fused to catalytically inactive Cas9 (dCas9) by sequencing the guide RNA (gRNA) barcodes associated with each peptide. We first confirm the ability of CasPlay to characterize antibody epitopes by recovering a known binding motif for a monoclonal anti-FLAG antibody. We then use a CasPlay library tiling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteome to evaluate vaccine-induced antibody reactivities. Using a peptide library representing the human virome, we demonstrate the ability of CasPlay to identify epitopes across many viruses from microliters of patient serum. Our results suggest that CasPlay is a viable strategy for customized protein interaction studies from highly complex libraries and could provide an alternative to phage display technologies.

摘要

蛋白展示技术将蛋白与独特的核酸序列（条形码）相连接，从而能够通过 DNA 测序对多重蛋白进行检测。在这里，我们开发了 Cas9 展示（CasPlay）技术，通过对与每个肽段相关的向导 RNA（gRNA）条形码进行测序，来检测融合了无催化活性 Cas9（dCas9）的定制化肽库。我们首先通过回收针对单克隆抗 FLAG 抗体的已知结合基序来证实 CasPlay 具有鉴定抗体表位的能力。然后，我们使用 CasPlay 文库对严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）蛋白质组进行平铺，以评估疫苗诱导的抗体反应性。通过使用代表人类病毒组的肽文库，我们证明了 CasPlay 能够从数微升的患者血清中鉴定出针对多种病毒的表位。我们的结果表明，CasPlay 是从高度复杂文库中进行定制化蛋白相互作用研究的可行策略，并且可能提供一种替代噬菌体展示技术的方法。