Sequencing data from Massachusetts General Hospital shows Cas9 integration into the genome, highlighting a serious hazard in gene-editing therapeutics

Abstract

The ability to edit a specific gene within our genomes using guided-nucleases (Cas9/ZFN/TALEN - CaZiTa) presents huge opportunities for curing many genetic disorders. Delivery of this ‘drug’ within cells is a critical step for such therapies. The ability of recombinant adeno-associated virus (rAAV) to enter cells makes it a perfect choice as a vector for gene therapy. A plasmid comprising the rAAV, the CaZiTa, guide RNAs (for CRISPR) is expected to enter the cell, edit the target gene(s), remain episomal, and thus fade away with time. However, the rather obvious danger of integration of the plasmid into the genome, if the episomal hypothesis is incorrect, is under-reported. A recent report has highlighted that bacterial genes from a plasmid were integrated into bovine genomes. Massachusetts General Hospital has recently published data on CRISPR edits (Accid:PRJNA563918), noting ‘high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks’. However, there is no mention of Cas9 integration. Here, the same data from Massachusetts General Hospital shows Cas9 integration in the exact edit sites provided for two genes - TMC1 and DMD. Also, there is a mis-annotation of one sample as ‘no gRNA’, since Cas9 integrations have been detected in that sample. This is an important distinction between AAV and CaZiTa integration: while AAV integration can be tolerated, Cas9 integration is a huge, and unacceptable, danger.

Cloning of human ABCB11 gene in E. coli required the removal of an intragenic Pribnow-Schaller Box before it’s Insertion into genomic safe harbor AAVS1 site using CRISPR–Cas9

“…“Targeted Integration and high transgene expression in AAVS1 Transgenic mice after in-vivo hematopoietic stem cell transduction with HDAd5/35++ Vectors” 28 is reported; however, to achieve this they used an adenoviral gene delivery system with AAV5 ITRs and AAV35 helper. Integration of AAV/ Cas9 into Cas9 mediated cut sites is a potentially hazardous consequence of this approach 29 , 30 .…”

The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing

“…These can be grouped as viral vectors, physical (i.e., microinjection or electroporation), or chemical methods, (cationic-lipid transfection and lipid nanoparticles), ligand fusion tags, cell-penetrating peptides, and gold nanoparticles, among others. The most common viral vectors are adenoviruses, adeno-associated viruses, and lentiviruses, since these are the ones approved for clinical trials (Chakraborty, 2019).…”

“…Viral vectors may lead to unintended Cas9 genome integration, raising important questions on the safety of clinical trials. This topic remains a matter of debate (Chakraborty, 2019). There is also the risk of immunogenicity both from the use of the virus and the expression of the Cas9 protein.…”

Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors

“…Moreover, gene editing cannot occur without the presence of both components in the same cell (i.e., the chance for a cell to receive two components is less than receiving one component). For therapeutic purposes, using plasmids may have potential safety concerns because introduced DNA may interfere with the host genome causing insertional mutagenesis [ 76 , 77 ]. A general consensus is that it is preferable to deliver the Cas nuclease as a functional protein because it saves the time during which the plasmid needs to be expressed; naked gRNA is relatively unstable and may get diffused or degraded, becoming unavailable to form a RNP complex with the nuclease when protein expression is completed [ 64 ].…”