| Objective: Better evaluation of the target and off-target effects of gene editing nucleases is highly useful. Here we report chromogenic assays for the evaluation of the target and off-target effects of CRISPR/Cas9. Both blue to white and white to blue assays have been developed based on the status of alpha complementation of the beta-galactosidase in E. coli. These new assays are particularly applicable in evaluating and selecting therapeutic gRNAs as the candidate gRNAs and candidate target sequences can be independently tested for the off-target effects.Methods: The targets of engineered CRISPR/Cas9 nuclease and the CRISPR/Cas9-gRNA were subcloned in different plasmids. To screen the off-target effects of the CRISPR/Cas9 in the present study, one set of targets with consecutive point mutations from a perfectly matched target sequence of angiotensinogen from the site 1 to 19 were constructed. Color changes of the colonies following co-transformation of these two plasmids can be visualized and the initial sensitive parameters in evaluating the target and off-target effects of the nuclease to be tested. To better quantitatively describe the effect following the gene editing by CRISPR/Cas9, a real time PCR was performed.To directly confirm the gene editing effects, either target or off-target effects, at the DNA sequence level an assay with white to blue color change was then designed by placing two repeats flanking the target to be tested. And another set of selected 4 targets with consecutive point mutation from the perfect target sequence of angiotensinogen from the site 1 to 19 were constructed, which is used for the white to blue assay.Results: When the targets and the relevant gRNAs are completely matched,virtually all colonies were white following the co-transformation, while the control plates were full with blue colonies. This blue to white color change of the colonies indicated that the CRISPR/Cas9 had its gene editing effect targeting the angiotensinogen gene and caused the loss or dramatically decrease of the beta-galactosidase activity in the E. coli following the co-transformation. However, fortargets with one-base mismatch, pale blue colonies were formed, indicating the off-target effects of the CRISPR/Cas9 occurred. For targets with two mismatched nucleotides that had high off-target effects at their source parent one-base mismatched targets, their combination showed a significantly lower off-target effects.The different degrees of blue to white color change may be resulted from varied off-target effects occurred in mismatched targets from those near the PAM site to ones far from the PAM site. According to the results of real time PCR,the gRNA matched with its target has the largest Ct1/2 and the not matched target control has the lowest Ct1/2, with mismatched targets having values between. Thus, the color change of the colonies formed following the co-transformation provides qualitative information about the gene editing occurred.The real time PCR can be used to quantitatively evaluate the efficiency of the gene editing enzymes.In the white to blue assays, targets to be tested are subcloned to cause a frame-shift effect to the beta-Gal gene, and thus white colonies are formed from the transformation of plasmids harboring these targets. The existence of these two repeats render the plasmid to possess the ability to have homologous recombination when the targets within the repeats are subjected to a double stranded break by gene editing with CRISPR/Cas9. Blue colonies are to be formed following the homologous recombination as the alpha peptide gene is in-frame again as designed. Different from the nearly homogenous change in blue to white colonies, there are some, but not all of the colonies,in blue color in the white to blue assay.Conclusion: The main purpose of the blue to white and white to blue assays developed in the present study is to provide practically useful technologies for the evaluation of the off-target effects of gene editing enzymes. |
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