Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in

Yi Jiun Chen, Ya Yun Cheng, Weikang Wang, Xiao Jun Tian, Daniel E. Lefever, David A. Taft, Jingyu Zhang, Jianhua Xing

Research output: Contribution to journalArticlepeer-review


Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing techniques find applications in many fields, such as molecular biology, cancer biology, and disease modeling. In contrast to the knock-out procedure, a key step of CRISPR knock-in experiments is the homology-directed repair process that requires donor constructs as repair templates. Therefore, it is desirable to generate a series of donor templates efficiently and cost-effectively. In this study, we developed a new strategy that combines (i) Gibson assembly reaction, (ii) a linker pair composed of eight in silico screened restriction enzyme sites, and (iii) a hierarchical framework, to remarkably improve the efficiency of producing donor constructs for common genes as well as for the genes containing unbalanced guanine-cytosine content and requiring a selectable marker. Furthermore, the approach provides the ability of inserting additional elements into the donor templates, such as single guide RNA recognition sites that have been reported to enhance the efficiency of homology-directed repair. Conclusively, our modularized process is simple, fast, and cost-effective for making donor constructs and benefits the application of CRISPR knock-in methods.

Original languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalBiology Methods and Protocols
Issue number1
StatePublished - 2021
Externally publishedYes


  • CRISPR knock-in
  • donor construct
  • in silico screening
  • molecular cloning

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)


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