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

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Long single-stranded DNA (ssDNA) is crucial for DNA nanotechnology, precision medicine, and CRISPR-Cas9 gene editing.
• Current methods for preparing long ssDNA are often inefficient, low-yield, and difficult to scale.
• There is a need for robust and scalable methods to produce high-purity long ssDNA.

Purpose of the Study:

• To develop and evaluate a novel workflow for the purification of kilobase-length single-stranded DNA (ssDNA).
• To demonstrate the scalability and reproducibility of the developed purification method.
• To assess the utility of the purified long ssDNA in genome engineering applications.

Main Methods:

• A workflow combining enzymatic digestion with high-temperature reversed-phase high-performance liquid chromatography (RP-HPLC) was developed.
• The method was validated using both analytical and semi-preparative formats.
• Purification efficiency was assessed for linear and circular ssDNA species ranging from 1.5 to 4.5 kilobases (kb).

Main Results:

• The RP-HPLC workflow achieved clean resolution of ssDNA species across various lengths and forms.
• The method demonstrated scalability across different purification formats.
• A 1.5 kb ssDNA donor template facilitated efficient CRISPR knock-in at the TRAC locus in human T cells without impacting cell viability or expansion.

Conclusions:

• The developed RP-HPLC workflow offers a scalable and reproducible method for producing high-purity long ssDNA.
• This method is suitable for generating ssDNA required for advanced genome engineering applications.
• The successful application in CRISPR-Cas9 demonstrates the practical utility of the purified long ssDNA.