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Open Access Publications from the University of California

Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies.

  • Author(s): Yang, Peng
  • Chou, Shih-Jie
  • Li, Jindian
  • Hui, Wenqiao
  • Liu, Wenfei
  • Sun, Na
  • Zhang, Ryan Y
  • Zhu, Yazhen
  • Tsai, Ming-Long
  • Lai, Henkie I
  • Smalley, Matthew
  • Zhang, Xinyue
  • Chen, Jiayuan
  • Romero, Zulema
  • Liu, Dahai
  • Ke, Zunfu
  • Zou, Chang
  • Lee, Chin-Fa
  • Jonas, Steven J
  • Ban, Qian
  • Weiss, Paul S
  • Kohn, Donald B
  • Chen, Kai
  • Chiou, Shih-Hwa
  • Tseng, Hsian-Rong
  • et al.

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate-mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)-encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

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