In our previous blogs on cell therapy solutions, we summarized the CRISPR-Cas (Clustered, Regularly Interspersed, Short Palindromic Repeats) system as a remarkable tool for greatly simplifying gene manipulation (insertions, deletions, development of knock-in and knock-out animal models). Also described were ways to study epigenetic processes by ‘borrowing’ the Cas9 nuclease from the CRISPR system to pair with histone acetylation domains for induction of acetylation at specific sites. For instance, one application of this capability could be applied in gene therapy, whereby incorporation of epigenetic changes could achieve gene silencing of abnormally activated genes, or by controlling stem cell differentiation.
Not surprisingly, at the most recent (2017) symposium of the American Society for Gene and Cell Therapy, use of the CRISPR –Cas system was prominently discussed as a viable gene delivery method including others such as engineered T cells (CAR-T), oncolytic viruses, Adeno Associated viruses and several others.
CRISPR-Cas9 Off-target effects
A key feature of the CRISPR –Cas 9 system is its ability to target DNA sequences of interest. This is accomplished by its integration of a guide RNA component that binds to complementary double-stranded DNA sequences. Once bound, the Cas 9 nuclease cleaves the DNA strands, creating a blunt ended DNA double stranded break, which can be repaired to yield insertions or deletions near the cleavage site, or deliberate DNA fragment insertion by supplying exogenous DNA sequences.
Interestingly enough, the initial targeting scrutiny of the CRISPR—Cas9 –guide RNA system revealed that not every nucleotide base in the guide RNA needed to be complementary to the target DNA sequence to effect Cas 9 nuclease activity. Further characterization of this phenomenon is needed, and Wu et al (1) summarized several methodologies that could be employed to characterize the binding domains and consequently Cas9 targeting specificity.
Situations where targeting of a region reveals identical copies of the target sequence in the genome has led to modification of the DNA at all of these sites when using CRISPR-Cas9. Similar situations have apparently occurred even when there is a single nucleotide mismatch between guide RNA and its complementary DNA target, based on the location of the mismatch in the guide sequence.
Recently (2016), Tsang et al (2) performed an in vivo CRISPR-Cas9 study to repair a genetic mutation in mice embryos that results in blindness. Sight restoration was achieved, but a number of off-target effects were also observed. When whole genome sequencing was performed on a CRISPR-Cas9 edited mouse, a large number of single nucleotide variants (SNVs) were found. None of these SNVs were found in any of the 36 strains in the Mouse Genome Project published sequence database, leading to the conclusion that the SNVs seen were the result of CRISPR-Cas9 off targeting. The authors concluded that at least certain guide RNAs may target loci independently of their target under in vivo conditions. Hence careful assay of guide RNA and Cas9 for off target mutations may be a critical first step prior to conducting actual gene therapy treatment.
In a related study, co-author Mahajan performed whole genome sequences on stored tissue samples from the 2016 work of Tsang et al and compared them to published Mouse Genomic DNA sequences, revealing that the CRISPR-Cas9 mice had ten times more mutations than expected, with none of these mutations present in the published data. Additionally, the computer algorithms used to predict mutation locations showed none of the locations identified by whole genome sequencing. Another outcome of the study suggested that conducting CRISPR-Cas9 experiments on cells in culture may not accurately predict how CRISPR-Cas9 editing will behave when the editing is done in whole organisms.
Other variables potentially include DNA sequence changes due to genetic drift in cells cultured in vitro as compared with the published genomic DNA sequences obtained from the organism, the predictive accuracy of algorithms currently in use for determining off-target effects, and a more comprehensive characterization of the CRISPR-Cas9 complex as it relates to in vivo targeting.
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(1) Target Specificity of the CRISPR-Cas9 System. Xuebing Wu, et al. Quant Biol June 2014; Vol 2(2)
(2) Unexpected mutations after CRISPR-Cas9 editing in vivo. Tsang et al. Nature Methods Jun 2017|. Vol 14 (6)pp.547-548
(3) CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa. Mahajan et al. Molecular Therapy August 2016 Vol.24 (8)pp.1388-1394