Using CRISPR/Cas9-based Gene Targeting to Study Cell Biological Processes
A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. For the past 25 years, the predominant strategy utilized in human tissue culture cells has been RNAi-mediated protein depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible protein depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells.
In this webinar, Dr. Iain Cheeseman of the Whitehead Institute for Biomedical Research will describe the strategies that his lab utilizes to analyze the cell biological phenotypes resulting from gene inactivation in human cells using Cas9-mediated gene knockouts.
First, he will discuss an inducible knockout strategy using modified cell lines with a doxycycline-inducible version of Cas9 and a stably expressed single guide RNA (sgRNA) introduced using a lentiviral vector. The inducible and conditional nature of the Cas9 induction allows a researcher to elucidate the consequences of both acute and chronic elimination for a target gene, which is particularly critical when analyzing genes required for cellular fitness, including essential genes. Using this strategy, the Cheeseman lab has generated and characterized a broad collection of inducible CRISPR/Cas9 knockout human cell lines targeting diverse cell cycle and cell division processes.
Second, Dr. Cheeseman will discuss alternate strategies for the effective introduction of the Cas9 guide RNA into target cells using synthetic guide RNA transfection. He will compare the efficiency of synthetic sgRNA and crRNA guides with that of cell lines stably expressing lentiviral-delivered RNA guides and will discuss technical considerations for effective use of synthetic guides for cell biological phenotypes.
Finally, Dr. Cheeseman will discuss strategies to conduct Cas9-based targeting for cell biological phenotypes at a larger scale, towards enabling genome-wide phenotypic screens. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.