Abstract
Recent data suggest that a time lag exists between nuclear penetration and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-directed gene editing activity in human cells. As CRISPR/Cas approaches clinical implementation, it is critical to establish a biological time frame in which the complex enters the cell and nucleus and executes its gene editing function. We are developing CRISPR-directed gene editing for the treatment of non-small cell lung carcinoma focusing Nuclear Factor Erythroid 2-Related Factor-Like (NRF2), a transcription factor which regulates chemoresistance. In this report, we define cellular events that surround the initialization of CRISPR-directed gene editing as a function of time. We analyze the efficiency of cellular transfection of both components of the RNP particle and assess the emergence of indels. For the first time, we image the nuclear positioning of the tracrRNA and Cas9 as a complex and as individual gene editing components. Our results indicate that while the nuclear localization of the CRISPR/Cas complex is efficient and rapid, disruption of the NRF2 gene appears four to eight hours later. We reveal an initial snapshot of the schedule and processing of CRISPR/Cas in a lung cancer cell; information that will be useful as cell-based protocols are designed and advanced.