Abstract
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.
Highlights
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas[9] system is widely used for various genome editing approaches in cultured cells and living organisms and was broadly explored for preclinical applications
To allow the use of multiple guide RNA (gRNA) expression units for multiplexing of the CRISPR/Cas[9] system, further gRNA expression units can be inserted into the shuttle plasmid
Additional gRNA expression units can be amplified via PCR using primers generating restriction enzyme sites for consecutive cloning of amplified gRNA expression units into respective restriction sites within pShV (Fig. 1B)
Summary
The CRISPR/Cas[9] system is widely used for various genome editing approaches in cultured cells and living organisms and was broadly explored for preclinical applications. AdV-mediated delivery was utilized to disrupt the HIV co-receptor in T-cells[10], to perform genome editing in adult murine liver[11] and for restoration of the dystrophin gene in vitro[12] and in vivo[13]. All of the latter studies applied early generation AdV vectors based on the human AdV type 5 deleted for the adenoviral early genes E1 and E3, which are known to cause anti-adenoviral innate and adoptive immune responses[14] suppressing effects of the viral vector. We evaluated this vector pipeline for delivery of gRNAs targeting clinically relevant targets including disruption of the HPV 18 genome, the HIV co-receptor CCR5, and the dystrophin gene affected in patients suffering from DMD
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