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News & Blogs » CRISPR News » Novel Approach for Improved Knock-In Efficiency in Primary Cells
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Novel Approach for Improved Knock-In Efficiency in Primary Cells

plague, FPR1, lentiCRISPR, black deathCombining the power of CRISPR technology and cell therapy, scientists are expediting their search for more effective and safer CAR T cell therapies. However, the CAR knock-in efficiency in human primary T cells may not be ideal yet, even with the most advanced ribonucleoprotein (RNP) and electroporation delivery method. This week, a study published in Nature Biotechnology suggested a novel approach that improves large gene insertion efficiency in primary cells by 2-6 fold1.

Previous study published last year in Nature by the same group in UCSF suggested that by employing CRISPR mediated homology directed repair (HDR) pathway, human primary T cells can be effectively and precisely engineered with the presence of a HDR template, for example single-stranded DNA (ssDNA)2. The integration of CAR into defined CRISPR cutting sites can be very well controlled and more precise when using ssDNA as the HDR template, with significantly reduced off-target integration by over 20 fold compared to dsDNA templates. Therefore, making engineered T cells much more accurate and safer.

However, CRISPR HDR based knock-in efficiency in these sensitive primary cells has not been very high. To address this issue, these scientists further explored methods that could improve the current efficiency. Their new study just published in Nature Biotechnology this week demonstrated a new approach in resolving this issue.

This new approach includes two improvements:

  • Adding truncated Cas9 target sequences (tCTSs) at each end of the homology-directed repair (HDR) template, so that these extended arms can interact with Cas9 RNPs and direct the template into the nucleus. This improvement alone is able to increase HDR insertion efficiency by 2-4 fold.
  • Using polyglutamic acid to stabilize Cas9 RNPs and form nanoparticles to further improve RNP delivery efficiency and knock-in rate by another 2 fold.

Together, this new method significantly enhanced HDR editing efficiency and knock-in efficiency in primary cells, bringing off-the-shelf CAR T cell therapy products even closer to reality.


1. Nguyen, D.N., Roth, T.L., Li, P.J. et al. Polymerstabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nat Biotechnol (2019) doi:10.1038/s41587-019-0325-6

2. Roth, T.L., Puig-Saus, C., Yu, R. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature559, 405–409 (2018) doi:10.1038/s41586-018-0326-5