CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5'-NNGRRT-3' PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells.

Genome editing using RNA‐guided nucleases in their ribonucleoprotein (RNP) form represents a promising strategy for gene modification and therapy because they are free of exogenous DNA integration and have reduced toxicity in vivo and ex vivo. However, genome editing by Cas9 nuclease from Staphylococcus aureus (SaCas9) has not been reported in its RNP form, which recognizes a longer protospacer adjacent motif (PAM), 5′‐NNGRRT‐3′, compared with Streptococcus pyogenes Cas9 (SpCas9) of 5′‐NGG‐3′ PAM. Here, SaCas9‐RNP‐mediated genome editing is reported in human cells. The SaCas9‐RNP displayed efficient genome editing activities of enhanced green fluorescent protein (EGFP) coding gene as well as three endogenous genes (OPA1, RS1, and VEGFA). Further, SaCas9‐RNP is successfully implemented to correct a pathogenic RS1 mutation for X‐linked juvenile retinoschisis. It is also shown that off‐target effects triggered by SaCas9‐RNP are undetectable by targeted deep sequencing. Collectively, this study demonstrates the potential of SaCas9‐RNP‐mediated genome editing in human cells, which could facilitate genome‐editing‐based therapy.

Edited by Ronald C. Wek CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNA Gln -processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5 nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA-sgRNA fusions improve Cas9 activity, we investigated whether a GN 20 sgRNA fused to different tRNAs (G-tRNA-N 20 ) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing-based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNA Gln -sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6-to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate highfidelity Cas9-mediated human genome-editing applications.CRISPR/Cas9, a type II system of CRISPR derived from the prokaryotic adaptive immune system, has great potential for genome editing and is under intense investigation at the present time (1, 2). The nuclease activity of Streptococcus pyogenes Cas9 (SpCas9), the most widely used nuclease at present, can be triggered by guide RNA targeting imperfectly matched off-target genomic sites. These off-target effects not only confound interpretation of results in the laboratory but also severely undermine the safety and reliability of clinical applications of the technology (3, 4). To address this issue, various strategies and efforts have been employed to minimize off-target activity, such as direct delivery (ribonucleoprotein complex) (5, 6), tunable systems (intein-inactivated Cas9, light-activated and small-molecule induction of Cas9) (7-9), separate Cas9 binding strategies (paired Cas9 nickases) (10), and truncated sgRNA (small guide RNA) 2 ( 11). With the delineation and optimization of Cas9 structure, high-fidelity Cas9 variants have been identified, including SpCas9-HF1, eSpCas9(1.1), HypaCas9, evoCas9, xCas9(3.7), .Recent studies revealed that sgRNA transcribed from the U3 or U6 promoter harboring an extra A or G(A/G) in the first nucleotide of the sgRNA may affect the activity of high-fidelity Cas9 variants (19, 20). Thus, selection of endogenous A/G in the first nucleotide position of the 20-nt target sequence is potentially useful. Additionally, different strategies have been adopted to address the extra nucleotide. The tRNA Gly from rice, expressed as a fusion with the guide sequence that would be processed by RNaseP and RNa...