Application Of Blue-White Colony Assay In The Off-target Evaluation Of CRISPR/Cas9

Objective: Better evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy.Here based on the blue-white screening technology,a prokaryotic evaluation system of the target and off-target effects of CRISPR/Cas9 has been established.This sensitive assay is particularly applicable in evaluating the off-target effects that caused by the mutation in PAM site and targets.Additionally,a white to blue colony assay is applied to the target effect evaluation for the gRNAs targeting EGFR mutation.Methods: To investigate the effects caused by mutation in PAM site,three sets of one to two nucleotides mismatched at PAM site were constructed into the LacZ region of PMD19-T vector to cause a in-frame effect.Under the X-Gal and IPTG induction,these vectors could generate blue bacterial colonies.After co-transformation of target and relevant gRNA vector,the cleavage activity of CRISPR/Cas9 could be observed through the color changes of colonies.To show the gene editing effects at the sequence level,a white to blue assay was designed by placing two HBV repeats flanking the targets to cause a frame-shift effect and their LacZ region could restore reading frame due to homologous recombination after edited by CRISPR/Cas9.By combining color changes with sequencing technology,the effects of CRISPR/Cas9 could be identified.To evaluate the off-target effects caused by different base mutations in target,seven sets of one to three nucleotides mismatched in target were subcloned into the LacZ region of PMD19-T to cause a in-frame effect.Using the prokaryotic evaluation system that had been established,we compared the target and off-target effects by the observation of color changes and analysis of blue-white ratio.Additionally,we apply the white-blue prokaryotic evaluation system to evaluate the target effects of the non-small-cell lung carcinoma EGFR 15 del.Three different PAM site targets,NGG,NGA,NAG respectively,were constructed.These targets were then subcloned into HBV repeats vector.After the co-transformation of these two vectors,we compared their efficiency by counting the number of blue colonies and analyzing sequencing results.Results: Co-transformation of completely mismatched plasmids and gRNAs yielded blue colonies while white colonies formed following co-transformation of the plasmids containing completely matched target and the gRNAs.We found that NAG could serve as a potential PAM site to maintain relatively high editing activities,but NNG or NGN could only display some gene editing activities.When there were two mutations in PAM site,they yielded dark blue colonies and their efficiency were only one fifth of NGG type.These data demonstrated the existence of the PAM site independent off-target effect.Blue colonies generated after co-transformation of the HBV repeats vectors containing relevant targets and the gRNAs.Sequencing results also identify the cleavage effects of CRISPR/Cas9.In the evaluation of the off-target effects caused by different base mutations in target,the colonies showed different degree of blue to white color change after the co-transformation of different targets and gRNAs.We recognized that the testing plasmid containing one base mismatch has significantly more off-target effect compared to that with two or three bases mismatch.When evaluating the target effects of the non-small-cell lung carcinoma EGFR 15 del,we found that the target containing NGG type PAM site had the highest efficiency.However,the efficiency of both NAG and NGA type targets decreased significantly due to the one base mutation in their PAM sites.Compared these two types,the efficiency of NAG type was obviously higher than that of NGA type,which was also identical with the results of part one.Conclusion: Based on blue-white screening technology,we developed a prokaryotic system to evaluate target and off-target effects of CRISPR/Cas9.Using the assay we developed,off-targets from the variable bases mismatched targets were confirmed and efficiency of gRNAs targeting EGFR mutation was compared.This confirmed phenomenon is informative for better understanding the severity of off-target effects in future human gene therapy.