Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing

The usefulness of genome editing lies in the ability to induce any desirable change in the target genome. It is most efficiently achieved using ssDNA (single-stranded DNA) as the HDR template and CRISPR-Cas9 targeted DNA breaks. However, the low efficiency of HDR integration is a challenge for achieving efficient edits. Among different HDR templates, ssDNA has the highest efficiency in inducing repair after CRISPR-induced DNA breaks. Several methods are used to generate the ssDNA-HDR template. However, each method has limitations in terms of feasibility for different ssDNA types, efficiency, time required, expenses incurred, etc. Often, these factors are overlooked, confusing users regarding the most appropriate method for generating ssDNA. This study describes and compares methods for generating ssDNA and outlines considerations for designing an efficient ssDNA template. Most frequently used methods are PCR-based, utilizing modified primers (phosphorylation, biotinylation, or phosphorothioate-based) or those utilizing the IVT-RT (In vitro transcription- reverse transcriptase) method. Asymmetric PCR and M13-based methods of ssDNA generation have been used as non-PCR methods. The IVT-RT method is widely adopted as it provides a middle-ground in yield and ease. The advantages and shortcomings of these methods, based on reported studies and our own results, are discussed.