Comparing CRISPR-Cas9 DNA Scissors
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Comparing CRISPR-Cas9 DNA Scissors – IBS Researchers

The most effective and convenient biotechnology tool used to cut specific DNA sequences is the CRISPR-Cas9. A multitude of variants have been engineered and employed for experiments worldwide, starting from Streptococcus pyogenes Cas9 (SpCas9). While all these systems are targeting and cleaving a specific DNA sequence, there are also potential harmful effects of these as they show relatively high off-target activities.

The most extensive high-throughput analysis of CRISPR-Cas9 activities has been achieved by the research team of the Center for Nanomedicine at the Institute for Basic Science (IBS, South Korea), led by Professor Hyongbum Henry Kim. The research team could predict the activities of SpCas9 variants for different DNA sequences by developing deep-learning-based computational models. A useful guide for selecting the most appropriate SpCas9 variant is represented in this study, published in Nature Biotechnology.

Schematics of the CRISPR-Cas9 system. A guide RNA guides Cas9 to the target DNA sequence, which is followed by the short protospacer adjacent motif (PAM). Researchers across the globe have been adopting this technology to cut DNA at desired positions. Credit: Kim, H., & Kim, J. S. Nature Reviews Genetics, 2014

Previous studies had evaluated only up to three

Cas9 systems, this study surpassed all the previous reports. 13 SpCas9 variants were compared by the IBS researchers and they defined which four-nucleotide sequences can be used as a protospacer adjacent motif (PAM) – a short DNA sequence positioned immediately after the DNA sequence targeted for cleavage that is required for Cas9 to cut.

The specificity of six different high-fidelity SpCas9 variants was evaluated additionally and was found that the original wild-type SpCas9 has the lowest specificity, while evoCas9 has the highest. At many target sequences, evoCas9 shows low activity although evoCas9 is very specific. These results suggest that high-fidelity Cas9 variants could be preferred depending on the DNA target sequence.

PAM compatibilities for SpCas9 variants. (a) Darker colors indicate a higher frequency of DNA cleavage. (b) Among these four variants (SpCas9, VRQR, xCas9, and SpCas9-NG), SpCas9-NG has been the traditional choice for all PAM sequences that have a guanine (G) as the second nucleotide. However, these results show that for PAM sequences AGAG and GGCG, for example, the Cas9 variant VRQR (in blue) would be preferable. Credit: Institute for Basic Science

A computational tool to predict the activities of SpCas9 variants, DeepSpCas9variants (deepcrispr.info/DeepSpCas9variants/), was developed by the IBS researchers based on these results. Users can take full advantage of the CRISPR technology, find out the most suitable SpCas9 variant, and may input the desired DNA target sequence by accessing this public website.

Comparing the specificity of the SpCas9 variants with a DNA sequence that has a single mismatch between the guide RNA and the target sequence. evoCas9 and the original SpCas9 exhibit the highest and the lowest specificity, respectively. Credit: Institute for Basic Science

Kim says, “A systematic comparison among the different SpCas9 variants was lacking and that’s when we started this research. The most appropriate SpCas9 variants can be selected by researchers for their own research purposes using DeepSpCas9variants.”

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Comparing CRISPR-Cas9 DNA Scissors

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