Applications of targeted CRISPR-Cas9 labeling with potential clinical diagnostic implications
In a recent publication, McCaffrey et al.describe the use of CRISPR-Cas9 technology for a new DNA labeling procedure, which can improve mapping of the human genome as well as analyses of complex genomic regions containing structurally variant and repetitive DNA that are typically not amenable to current labeling methods.
They demonstrate the use of a mutant form of Cas9, which is characterized as a Cas9 nickase (Cas9n), to nick DNA complementary to crRNA. This mutant is has been previously used to precisely target opposite strands of the same locus to generate double-strand breaks (DSBs); however, in this paper theCas9n-dependent nicking protocol is adapted further to fluorescently label specific sequences for whole-genome mapping through in vitro nick-labeling. Appropriate restriction site motifs are often missing from repetitive regions of the genome, making analysis difficult, but this new method can map the breaking points of structural variations by directing labeling near these breakpoints via CRISPR-Cas9 guide RNAs (gRNAs).
Use and Advantages of Edit-R Synthetic Guide RNAs
Synthetic crRNAs and universal tracrRNA from Dharmacon, Inc. were used in order to create specific gRNAs to be incorporated into the nick-labeling procedure. This approach involved precise nicking and fluorescent labeling of sequences in one color, followed by labeling in a second color via the global nickase enzyme motif. In addition to synthetic guide RNAs from Dharmacon, vector-expressed single guide RNAs (sgRNA) were also tested. In both cases, the Cas9n made a targeted cut in one strand of the target double-strand DNA, three nucleotides upstream of a protospacer adjacent motif (PAM), and fluorescently labeled nucleotides were directly incorporated,. However; the labeling efficiencies of the synthetic gRNAs were found to be much higher than those achieved using sgRNAs.
While cloning and sequence verification of sgRNA vectors can be laborious and time consuming, and in vitrotranscription of sgRNA requires additional time and quality control to ensure consistency in length and purity of the transcribed product, the Dharmacon approach of chemical synthesis for rapidly generating crRNA and tracrRNA molecules separately has clear time and effort advantages for any gene editing workflow. This is shown with the application of synthetic guide RNAs to the approach in the paper by McCaffrey et al., which promises to dramatically improve DNA mapping in complex and structurally variant genomic regions, as well as facilitate high-throughput automated whole-genome mapping.
Conclusions
This study demonstrates a new application of Edit-R guide RNAs for use in next-generation genome mapping and structural analysis. The newly described approach in this study allows for mapping of specific sequence information along long complex DNA molecules and makes the mapping of the human genome more accurate, but it can also be used to specifically target certain loci for clinical testing. The clinical implications as well as the ease of use of synthetic guide RNAs as part of the CRISPR-Cas9 adapted approach will undoubtedly prove to be yet another valuable new application of gene editing to improving the human condition.
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