December 2019

Improved Cas9 activity by specific modifications of the tracrRNA

Tristan Scott, Ryan Urak, Citradewi Soemardy & Kevin V. Morris

CRISPR/Cas9 is a breakthrough technology which allows scientists to modify genome as intended. It has a wide range of application in many different fields including improvement of biotechnology products, fundamental biological research and treatment of diseases. It has already demonstrated promising results for therapeutic applications; however the technology still needs to be improved to avoid any unintended modifications. For this purpose, scientists have been developing different approaches to increase the CRISPR efficiency and precision, such as using fusion proteins, modifying sgRNA, length reduction of crRNA. However, not many researches have focused on modification of tracrRNA.

In this paper, the research group has demonstrated that modified tracrRNA can improve reporter knockdown and indel formation at several target sites and it can be used for research as well as therapeutic applications. CRISPR/Cas9 required crRNA and tracrRNA for its activity. These two molecules can be combined either through a direct repeat sequence to function as a dual-guide RNA (dgRNA) or through a tetraloop to form a small-guide RNA (sgRNA). In both cases, they can be preloaded to Cas9 to form Cas9-RNP complex. In this study, they showed that specific modifications on tracrRNA can significantly increase Cas9-RNP activity. To do so, they have modified the tracrRNA including several different modifications (replacement and/or deletion of uridines) followed by screening of U-modified tracrRNAs by comparing Cas9-knockdown activity on human immunogenicity virus (HIV) reporter cell lines. As a result of this they identified specific sequence modification to the gRNA that significantly enhance Cas9 activity.

Firstly, they aimed to show the importance of uridines on tracrRNA sequence. Focusing on the replacement of uridines with other nucleotides (adenines and guanines), they demonstrated that chemically uridines modified tracrRNAs are resulted in a loss of Cas9 activity. Keeping that information in mind, they screened seventeen different U-modified tracrRNAs and they have selected three specific modifications resulting in increase in knockdown activity based on relative inactivation of GFP activity in pMoHIV-C6¹ cells. They also verified the efficiency at genomic level by analyzing Sanger sequencing data on TIDE². They have also examined whether Cas9 activity can be improved further. Thus, they modified and combined these top-three U-modified tracrRNAs and compared their efficiencies. The results have showed that one of the combined tracrRNA is yielded in increased efficiency compared to unmodified and single-modified tracrRNAs. After testing different modifications at one target site with specific crRNA, they have demonstrated that sequence modified tracrRNA can also improve Cas9-RNP activity with alternative crRNAs at same target site as well as at different genomic target site.

In addition, they also wanted to demonstrate that this approach is also efficient on primary cells as well as cell lines, so they isolated CD4+ T cells from human donors and screened the efficiency by measuring the reduction in CCR5 surface marker after gene editing.

Although additional studies are needed to validate the effects of this approach, they show that it can be efficient with different crRNAs targeting same site, in other cell types and/or at additional target sites. When they identified the modifications resulting in increased Cas9 activity, they realized that these modifications particularly located in the linker region and stem-loop in the tracrRNA. Besides, they also compared the efficiency of gRNA when they are diluted, and they demonstrated that reduced amount of U-modified tracrRNA could result in comparable Cas9 efficiency when compared to unmodified tracrRNA which is an important advantages especially for gene therapy and clinical use. Due to the variation in activity with specific crRNAs in different cells and indel formation, they suggested that further validation is needed to better understand the conditions that give rise to improve activity. To sum up, they highlighted that this novel and easy modified tracrRNA can be combined with current technology to improve CRISPR/Cas9 efficiency, especially for clinical applications

¹ a clonal HEK293 cell line with a LTR driving high levels of GFP expression

² a web tool which quantifies editing efficacy and identifies predominant types of indels.