Improved Cas9 activity by specific modifications of the tracrRNA.

CRISPR/Cas is a transformative gene editing tool, that offers a simple and effective way to target a catalytic Cas9, the most widely used is derived from Streptococcus pyogenes (SpCas9), with a complementary small guide RNA (sgRNA) to inactivate endogenous genes resulting from insertions and deletions (indels). CRISPR/Cas9 has been rapidly applied to basic research as well as expanded for potential clinical applications. Utilization of spCas9 as an ribonuclearprotein complex (RNP) is considered the most safe and effective method to apply Cas9 technology, and the efficacy of this system is critically dependent on the ability of Cas9 to generate high levels of indels. We find here that novel sequence changes to the tracrRNA significantly improves Cas9 activity when delivered as an RNP. We demonstrate that a dual-guide RNA (dgRNA) with a modified tracrRNA can improve reporter knockdown and indel formation at several targets within the long terminal repeat (LTR) of HIV. Furthermore, the sequence-modified tracrRNAs improved Cas9-mediated reduction of CCR5 surface receptor expression in cell lines, which correlated with higher levels of indel formation. It was demonstrated that a Cas9 RNP with a sequence modified tracrRNA enhanced indel formation at the CCR5 target site in primary CD4+ T-cells. Finally, we show improved activity at two additional targets within the HBB locus and the BCL11A GATA site. Overall, the data presented here suggests that novel facile tracrRNA sequence changes could potentially be integrated with current dgRNA technology, and open up the possibility for the development of sequence modified tracrRNAs to improve Cas9 RNP activity.