Single-stranded DNA (ssDNA or ssODN) is proven to be a reliable CRISPR homology directed repair (HDR) template for creating gene knock-in with high editing efficiency, minimal toxicity, and reduced off-target integration.

The newly designed hybrid ssDNA HDR templates (HDRTs) that incorporate Cas9 target sequences (ssCTS) enabled high knock-in efficiencies (>60%) across a variety of target loci, knock-in constructs, and primary human cell types.

At a clinical scale for non-viral CAR-T cell manufacturing, ssDNA HDRTs enabled high knock-in efficiencies of ~46%.


The CRISPR/Cas9 genome editing toolkit has been widely used and adapted for diverse applications due to its simplicity and versatility. Its competence to offer precise and efficient genome editing makes it a powerful research tool for both knocking out endogenous TCR and inserting functional chimeric antigen receptor (CAR) or T cell receptor (TCR) for developing next-generation CAR/TCR-T cells.

Both viral and non-viral methods for delivery of CRISPR/Cas9 gene-editing tools and payload templates have been recently developed. Viruses have been traditionally used for delivering knock-in gene templates into cells with high efficiency, yet issues including high immunogenicity, increased risk of insertional mutagenesis, high cost and long lead time for manufacturing have raised concerns among researchers. Non-viral delivery of CRISPR/Cas9 editing tools and payload template DNA holds advantages by enabling site-specific transgene insertion via homology-directed repair (HDR) with minimized toxicity and reduced regulatory concerns. More and more researchers are trying to improve the efficiency of non-viral methods to develop a safer and more effective approach for gene and cell therapy1.

Dr. Nguyen and Roth et al. previously reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDR templates (HDRTs) to improve knock-in efficiency. In this approach, CTS allow the co-electroporated ribonucleoprotein (RNPs) to bind the HDRTs, facilitating their delivery into the nucleus and increasing insertion efficiency by bringing knock-in templates close to CRISPR RNP cutting sites2. Yet the yields and efficiencies were limited by toxicity at higher dsDNA concentrations.

In this case study, Dr. Shy et al. developed a hybrid single-stranded DNA ssDNA HDRTs with CTS (ssCTS), which boosted knock-in efficiency by >5-fold and the live cell yields by >7-fold. This strategy takes advantage of ssCTS's low toxicity at higher concentrations compared to dsDNA HDRTs with CTS (dsCTS). Due to the challenge in the production of long ssDNA sequences, Dr. Shy et al. collaborated with GenScript and were able to perform therapeutic gene editing on primary T cells at a clinical scale3


  1. Ehsan Razeghian et al. A deep insight into CRISPR/Cas9 application in CAR-T cell-based tumor immunotherapies. Stem Cell Research & Therapy, 428 (2021) .
  2. Nguyen, D. N. et al. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nature biotechnology 38, 44-49, doi:10.1038/s41587-019-0325-6 (2020).
  3. Brian Shy et al., Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing. bioRxiv, Sep 2021.

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