(176d) Simple and Efficient Gene Knock-in Strategy in Human Cell Lines Using 5’ Modified dsDNA Donors with Short Homology Arms

Yi Yu^1,#, Yijun Guo^1,2,#, Qiqi Tan^4,#, Yuanqing Lan⁴, Hugh Yeh³, Meng Zhang¹, Ipek Tasan⁴, Surbhi Jain⁴, Huimin Zhao^1,4,5,6,*

¹Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801

²Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, China, 230026

³School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801

⁴Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801

⁵Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801

⁶Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801

^# These authors contributed equally to this work.

*To whom correspondence should be addressed. Phone: (217) 333-2631. Fax: (217) 333-5052. E-mail: zhao5@illinois.edu

Abstract

CRISPR/Cas9-induced double-strand breaks (DSBs) enable various genome editing applications including gene knock-out (KO), gene knock-in (KI), and introduction of point mutations. Gene KI has been explored for a wide variety of purposes ranging from functional genomics to gene therapies. Despite its enormous potential in biotechnology and pharmaceutical industry, gene KI is very inefficient in human cells, especially in stem cells (typical gene KI rate in human stem cells is lower than 0.1% without selection). Recent efforts to increase the efficiency of CRISPR/Cas9-mediated gene KI in human cells include enhancing Cas9 cleavage, optimizing donor templates design, and supplementing small molecules to promote homologous recombination (HR). However, gene KI efficiency is still far from what is needed for complete editing of cells and varies greatly at different genomic loci and among cell types (typical gene KI rate ranging from 0.001% to 30% for >500 bp insertion). Due to the paucity of cells containing the desired edits, successful gene KI often requires weeks of post-treatment selections.

In this work, we developed a rapid CRISPR/Cas9-mediated gene knock-in strategy that utilizes Cas9 ribonucleoprotein and 5’ modified dsDNA donors with 50 bp homology arms and achieved unprecedented 65%/40% KI rate for 0.7 Kb/2.5 Kb insert in HEK293T cells. The identified 5’ end modification led to up to 5-fold increase in gene KI rates at various genomic loci in human cancer and stem cells. In addition, by decreasing the time required for gene KI to 1 day, this method is potentially useful in high-throughput cell line engineering.