Intramolecular interactions in gRNA may influence Cas9 cleavage activity

Bill Reid, Ph.D. Postdoctoral Researcher, Institute for Bioscience and Biotechnology Research, University of Maryland College Park

Bill Reid, Ph.D. Postdoctoral Researcher, Mosquito and Fly Research Unit, USDA/ARS, Center for Agricultural, Medical and Veterinary Entomology, Gainesville, Florida MORE ABOUT THE AUTHOR

In an article recently published in Nature Communications, Thyme et al (2016) discuss the interactions of the sequences of sgRNAs, the role of chromatin, and how these factors might interfere with Cas9 cleavage activity.
By investigating the activity of Cas9 cleavage both in vitro and in vivo (zebrafish embryos), they found that gRNA identified as highly active in vitro did not necessarily have high activity in vivo.

The CTCF protein functions as an insulator protein and is involved in the regulation of chromatin structure. The binding of CTCF occurs throughout the genome as shown above (green bands) on these polytene chromosomes of D. melanogaster (Mohan et al., 2007).

Upon closer examination, they found that genomic sites containing putative CTCF binding motifs yielded low Cas9 cleavage, while neighboring sites (within 100bp) displayed high activity.

In addition to finding a correlation connecting low Cas9 activity with CTCF binding motifs, they investigated the role of sequence complementarity within sgRNAs – specifically examining the influence of changing the 20nt gRNA that targets the DNA.

Predicted hairpins within the 20nt gRNA that targets the genomic DNA cleavage site resulted in reduced Cas9 cleavage activity. The predicted structure on the left represents an inactive gRNA, the predicted structure on the right shows an active gRNA.

Through secondary structure prediction, they found that the presence of hairpins predicted to be strong in the 20nt gRNA correlated with low Cas9 activity, and further demonstrated through nucleotide substitutions that strong internal gRNA interactions resulted in decreases in Cas9 cleavage activity.

Similarly, they identified that similar interactions between the 20nt gRNA and the scaffold portion of the sgRNA reduced Cas9 cleavage activity, and that this could be alleviated when complementary substitutions were made in the scaffold portion to remove the hairpin interaction with the gRNA, or if the sgRNAs were denatured with heat and allowed to refold.

In vitro synthesized sgRNA showing the intra/inter-molecular interactions within the sgRNA: A) NEB 100 bp ladder, B) DNA template for sgRNA synthesis, C) sgRNA after synthesis, D) same sgRNA sample denatured for 2 min at 70°C then placed on ice for 1 min (500 ng loaded per lane).

While predicted hairpins were not associated with decreased Cas9 activity in 100% of the guides tested, the correlation was strong enough for the authors to develop a predictive model to scan gRNAs for potential internal interactions (as a stand-alone Python script), and have also incorporated this predictive tool in the online version of CHOPCHOP.

This study highlights how the chromatin structure and the predicted secondary structure of the sgRNA could influence the activity of Cas9 cleavage; factors worth considering when designing sgRNAs and preparing an injection mastermix.
Thyme SB, Akhmetova L, Montague TG, Valen E, Schier AF. 2016. Internal guide RNA interactions interfere with Cas9-mediated cleavage. Nature Communications. DOI:10.1038/ncomms11750.



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