[PMC free article] [PubMed] [Google Scholar] 26. has been the most highly used CRISPR system and its use for genome editing only requires the presence of the Cas9 nuclease and a guide RNA (gRNA). In concern for translation of this methodology to clinical trials. While several groups have described novel mutant Rabbit Polyclonal to ADCK5 Cas9 enzymes with reduced off-target cleavage activity26C28, these experiments were performed with plasmid-based Cas9 delivery several groups have described novel mutant Cas9 enzymes with reduced off-target cleavage activity26C28, these experiments were performed with plasmid-based Cas9 delivery systems in immortalized cell lines, as opposed to RNP delivery. Therefore, discovery of a Cas9 mutant that does not sacrifice on-target activity while reducing OTEs in the RNP context would have great impact on therapeutic genome editing. Using an unbiased bacterial screening approach, we identified a single point mutation (R691A) in Cas9 (hereafter referred to as high fidelity Cas9, or HiFi Cas9) that reduces global OTEs while maintaining high on-target activity when used as an RNP complex. When compared to the R691A HiFi Cas9, we demonstrate that this rationally-designed eSpCas9(1.1)26, SpCas9-HF127, and HypaCas928 high-fidelity Cas9 mutants suffer reduced on-target editing at many sites when used as an RNP. We demonstrate clinical power of HiFi Cas9 in targeting several important disease-associated loci for HR in clinically-relevant primary human CD34+ HSPCs and T-cells. We also show robust correction of the sickle cell disease (SCD)-causing Glu6Val mutation in HSPCs, while reducing OTEs up to 20 fold compared to wild-type (WT) Cas9. RESULTS Existing Cas9 mutants with improved specificity also exhibit reduced on-target activity with RNP delivery While delivery of the Cas9:gRNA complex as a RNP can dramatically reduce OTEs, certain guideline sequences still cleave off-target sites regardless of ALS-8112 the delivery mechanism15,16,29. We compared the relative on- and off-target cleavage activities using RNP delivery in HEK293 cells for WT Cas9 and two published mutants, eSpCas9(1.1) (K848A, K1003A, and R1060A) or SpCas9-HF1 (N497A, R661A, Q695A, and Q926A)26,27 at three previously characterized guideline sites, (Figs. 1a and ?and1b).1b). The eSpCas9(1.1) mutant showed an on-target editing efficiency that was similar to WT Cas9 with the guideline; however, it only functioned at 60% of WT when using both the or guides. The SpCas9-HF1 protein had even lower activity, showing 28% of WT with the guide and 12% of WT with the guide (Fig. 1a,b). A reduction in Cas9 activity for SpCas9-HF1 and eSpCas9(1.1) delivered as a RNP has been previously described, ALS-8112 confirming the disadvantage of using these engineered Cas9 variants in the RNP format for high efficiency gene editing16,30. On-target editing activity in RNP format was further tested using 9 guides that target sites within ALS-8112 the human genes. The eSpCas9(1.1) mutant on average produced only 23% of the WT Cas9 editing activity, with the best guideline in this set showing 56% of the WT activity. The SpCas9-HF1 mutant showed even lower performance and on average produced only 4% of the WT Cas9 editing activity, with the best guide in this set showing 12% of the WT activity (Fig. 1c). Thus these mutants, both of whom bear multiple amino acidity changes, display a pronounced decrease in on-target activity when used in combination with short length RNP delivery. Open up in another window Shape 1 On-target activity of high-fidelity Cas9 mutants in human being cells with ribonucleoprotein (RNP) delivery.(a) Editing efficiency from the WT (blue), eSpCas9(1.1) (orange), or SpCas9-HF1 (grey) Cas9 proteins with crRNAs that focus on loci in HEK293 cells. The on-target site loci in HEK293 cells. Pubs represent suggest s.e.m., bacterial testing methods have already been adapted to.