A Site-Specific Integration Reporter System that Enables Rapid Evaluation of CRISPR/Cas9-mediated Genome Editing Strategies in CHO Cells.

Targeted gene knockout (KO) and site-specific integration (SSI) are powerful genome editing techniques to improve the development of industrially relevant Chinese hamster ovary (CHO) cell lines. However, past efforts to perform SSI in CHO cells have been characterized by low efficiencies, necessitating tedious workflows to obtain a clone in which precise, rather than random, transgene integration has occurred. Moreover, numerous strategies have been proposed to boost SSI efficiency in mammalian cell types, yet few have been evaluated head to head or in combination to appreciably boost efficiencies in CHO. To enable systematic and rapid optimization of genome editing methods, we present the SSIGNAL ( site- specific integration and ge nome alteration) reporter system, a tool which can be operated in either of two modes to analyze CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated disruption activity alone, or in conjunction with SSI efficiency. The reporter system functions by using green and red dual-fluorescence signals to indicate genotype states within four days following transfection, facilitating rapid data acquisition via standard flow cytometry instrumentation. In addition to describing the design and development of the system, we demonstrate two of its applications by first comparing transfection conditions to maximize CRISPR/Cas9 activity and subsequently assessing the efficiency of several promising SSI strategies. Due to its sensitivity and versatility, the SSIGNAL reporter system may serve as a tool to advance genome editing technology. This article is protected by copyright. All rights reserved.