14 December 2020

November 2020


Highly Parallel Profiling of Cas9 Variant Specificity

CRISPR-Cas9 technology has been closely investigated to increase its efficiency and accuracy since its discovery. There have been many different approaches which are aiming to improve this powerful gene-editing tool. Although CRISPR technology is a powerful gene-editing technique, there is still a need for optimization of mechanism to use it as a risk-free (as much as possible) therapeutic tool. When a double-strand break (DSB) is introduced to a target site on the host genome by Cas9, there is a possibility of inserting or deleting nucleotides (indels), so-called off-target activity. Off-target outcomes are highly dependent on the guide RNA and also nuclease protein. Thus, it is important to determine empirically the interaction between these two components of CRISPR and the possible off-target outcomes. Currently, the majority of CRISPR experiments are based on S. pyogenes Cas9 (SpCas9) cleavage, however, scientists are continuously testing different Cas9 species including both engineered or evolved variants. In this paper, the authors developed a novel approach called tagmentation-based tag integration site sequencing (TTISS) to assess editing outcomes and also used this method to screen different SpCas9 variants to profiling them in terms of their specificity and activity scores.

TTISS off-target detection method relies on identification of double-strand breaks by sequencing. A simplified method of this technique starts with tagging DSBs through the integration of a double-stranded donor DNA exploiting guide multiplexing and bulk tagmentation by Tn5. It can be used directly in lysed cells which provide a rapid and easy protocol. After tagmentation, DNA is isolated by a spin column. It is then followed by an enrichment of tagged sequence by two nested PCRs to obtain a final product. Then, sequenced integration sites are listed and off-target sites for each guide and Cas9 protein can be determined.

In the result part, they have tested the interaction between the activity and the specificity of SpCas9 variants by using TTISS method. After analyzing this tool in nine different SpCas9 variants, based on their findings they concluded that LZ3 Cas9 shows increased activity and also specificity as compared to WT SpCas9. In conclusion, this novel tool provides an accessible, rapid, cost-effective and scalable experimental setup for determining off-targets and +1 insertion frequencies after CRISPR/Cas9 genome editing.