RP-HPLC-based purification of long single-stranded DNA for CRISPR knock-in applications.

IF 2.5 4区工程技术 Q3 BIOCHEMICAL RESEARCH METHODS

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-04-09 DOI:10.1080/07366205.2026.2649857

Justina Martinkienė, Tingting Cui, Giovanni Ciotta, Juozas Kupčinskas, Deividas Pažėraitis

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引用次数: 0

Abstract

BackgroundLong single-stranded DNA (ssDNA; >200 nucleotides) is valuable for DNA nanotechnology, precision medicine, and as a CRISPR-Cas9 knock-in donor template, but existing preparation methods are laborious, low-yield, or difficult to scale. MethodsWe developed a workflow combining enzymatic digestion with high-temperature reversed-phase high-performance liquid chromatography (RP-HPLC) to purify kilobase-length ssDNA. The method was evaluated across analytical and semi-preparative formats. ResultsThe approach enables clean resolution of linear and circular ssDNA species ranging from 1.5 to 4.5 kb and is scalable across formats. A 1.5 kb ssDNA donor supported efficient CRISPR knock-in at the T-cell receptor alpha constant (TRAC) locus in primary human CD8⁺ T cells, without adversely affecting viability or expansion. ConclusionsThis RP-HPLC workflow provides a scalable and reproducible method for generating high-purity long ssDNA suitable for genome engineering applications.

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用于CRISPR敲入应用的长单链DNA的rp - hplc纯化。

长单链DNA （ssDNA，约200个核苷酸）在DNA纳米技术、精准医学和CRISPR-Cas9敲入供体模板方面具有重要价值，但现有的制备方法费力、产量低或难以规模化。方法建立了酶切与高温反相高效液相色谱（RP-HPLC）相结合的纯化千碱基长度ssDNA的工作流程。该方法被评估跨分析和半制备格式。结果该方法能够清晰地分辨1.5 ~ 4.5 kb的线性和圆形ssDNA物种，并具有跨格式的可扩展性。一个1.5 kb的ssDNA供体支持原代人CD8 + T细胞中T细胞受体α常数（TRAC）位点的高效CRISPR敲入，而不会对生存能力或扩增产生不利影响。结论该反相高效液相色谱流程为高纯度的长链单链dna的制备提供了一种可扩展、可重复的方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

BioTechniques 工程技术-生化研究方法

CiteScore

4.40

自引率

0.00%

发文量

审稿时长

3.3 months

期刊介绍： BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies. Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers: Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices Surveys of technical approaches related to broad fields of research Reviews discussing advancements in techniques and methods related to broad fields of research Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.