RT Journal Article
SR Electronic
T1 Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 219618
DO 10.1101/219618
A1 Yi-Jiun Chen
A1 Weikang Wang
A1 Xiao-Jiun Tian
A1 Daniel E. Lefever
A1 David A. Taft
A1 Jianhua Xing
YR 2017
UL http://biorxiv.org/content/early/2017/11/14/219618.abstract
AB CRISPR-based gene knock-in at endogenous sites is desirable in multiple fields such as quantitative studies of signal transduction pathways and gene regulation, synthetic biology, and disease modeling. Contrasting the knock-out procedure, a key step of CRISPR knock-in procedure relies on the homology-directed repairing (HDR) process that requires a donor construct as repair template. Therefore, it is desirable to generate a series of donor DNA constructs efficiently and cost-effectively. In this study, we developed a general Gibson assembly procedure that combines strengths of a Modular Overlap-Directed Assembly with Linkers (MODAL) strategy and a restriction enzyme based hierarchical framework. This procedure also allows fusing sgRNAs to the constructs for enhanced homology-directed repairing efficiency. Experimental tests on multiple constructs achieved from 3-8 folds of increase in assembly efficiency to high yield of constructs that failed to make with conventional Gibson assembly. The modularized procedure is simple, fast and cost-effective while making multiple constructs, and a computer package is provided for customized design.