Li F, Hung SS, Wang S, Lim JK, Wang JH, Tu L, Daniszewski M, Lo C, Wong RC, Pébay A, King AE, Bui BV, Liu GS, Hewitt AW.
Delivery of CRISPR/Cas proteins remains one of the major remaining impediments to the widespread application of in vivo gene editing and the anticipatory cures of monogenic retinal diseases. We recently reported the utility of viral mediated CRISPR/Cas gene editing in the retina; however, with such a viral delivery system, active exogenous endonucleases will be maintained in the retina for an extended period. Consequently, CRISPR/Cas genotoxicity is a significant consideration in clinical applications. To address this issue, we have rationally designed a self-destructing “kamikaze” CRISPR/Cas system that disrupts the CRISPR/Cas enzyme itself after active protein expressed. Four different guide RNAs (sgRNAs) were designed to target Streptococcus pyogenes Cas9 (SpCas) and after in situ validation, selected sgRNAs were cloned into the pX552-YFP-sgRNA plasmid to generate yellow fluorescent protein (YFP) targeting “kamikaze-CRISPR/Cas” vector (pX552-YFP sgRNA-SpCas9 sgRNA). The editing efficacy our “YFP targeting kamikaze-CRISPR/Cas” constructs were further validated in vitro in YFP expressed HEK293 cells by Western blot and FACS analysis. Constructs were packaged into an adeno-associated virus 2 (AAV2) vector and administered intravitreally in the Thy1-YFP transgenic mouse and the expression of spCas9 as well as number of YFP fluorescent cells in the retinas were evaluated at 8 weeks after injection. AAV2-mediated LacZ or single YFP targeting SpCas9 sgRNAs were used as controls. Marked reduction of SpCas9 protein concentration (90% at day 2) as well as YFP expression (~66% to 80% at day 10) was found following transfection of our YFP targeting kamikaze-CRISPR/Cas vector in YFP-expressing HEK293 cells compared to the LacZ targeting CRISPR/Cas control. AAV2-mediated in vivo delivery of this construct significantly reduced the number of YFP fluorescent cells (~86% reduction) in the inner retina of Thy1-YFP transgenic mice. Moreover, a significantly decrease of spCas9 mRNA was also detected in AAV2-mediated YFP targeting kamikaze-CRISPR/Cas infected retina over relative to those treated with YFP targeting CRISPR/Cas alone. CRISPR/Cas genotoxicity is a significant consideration in future clinical applications. Our work has demonstrated that a “self-destructing/kamikaze” CRISPR/Cas can be used as a safe and robust tool for refined gene editing in the retina.