Optimized CRISPR/Cas9 strategy for homology-directed multiple targeted integration of transgenes in CHO cells

Biotechnol Bioeng. 2020 Jun;117(6):1895-1903. doi: 10.1002/bit.27315. Epub 2020 Mar 2.


Site-specific integration has emerged as a promising strategy for precise Chinese hamster ovary (CHO) cell line engineering and predictable cell line development (CLD). CRISPR/Cas9 with the homology-directed repair (HDR) pathway enables precise integration of transgenes into target genomic sites. However, inherent recalcitrance to HDR-mediated targeted integration (TI) of transgenes results in low targeting efficiency, thus requiring a selection process to find a targeted integrant in CHO cells. Here, we explored several parameters that influence the targeting efficiency using a promoter-trap-based single- or double-knock-in (KI) monitoring system. A simple change in the donor template design by the addition of single-guide RNA recognition sequences strongly increased KI efficiency (2.9-36.0 fold), depending on integration sites and cell culture mode, compared to conventional circular donor plasmids. Furthermore, sequential and simultaneous KI strategies enabled us to obtain populations with ~1-4% of double-KI cells without additional enrichment procedures. Thus, this simple optimized strategy not only allows efficient CRISPR/Cas9-mediated TI in CHO cells but also paves the way for the applicability of multiplexed KIs in one experimental step without the need for sequential and independent CHO-CLD procedures.

Keywords: CRISPR/Cas9; Chinese hamster ovary (CHO); double cut donor; homology-directed repair; multiple knock-in; targeted integration.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Cricetulus
  • Gene Knock-In Techniques / methods*
  • Plasmids / genetics
  • RNA, Guide, Kinetoplastida / genetics
  • Transgenes*


  • RNA, Guide