The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without killing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.

The human pathogen Pseudomonas aeruginosa uses N-butyryl-L-homoserine lactone (BHL) and N-(3-oxododecanyl)-L-homoserine lactone (OdDHL) as small molecule intercellular signals in a phenomenon known as quorum sensing (QS). QS modulators are effective at attenuating P. aeruginosa virulence; therefore, they are a potential new class of antibacterial agent. The lactone in BHL and OdDHL is hydrolysed under physiological conditions. The hydrolysis proceeds at a rate faster than racemisation of the alpha-chiral centre. Non-hydrolysable, non-racemic analogues (small molecule probes) were designed and synthesised, replacing the lactone with a ketone. OdDHL analogues were found to be relatively unstable to decomposition unless they were difluorinated between the beta-keto amide. Stability studies on a non-hydrolysable, cyclohexanone analogue indicated that racemisation of the alpha-chiral centre was relatively slow. This analogue was assayed to show that the L-isomer is likely to be responsible for the QS autoinducing activity in P. aeruginosa and Serratia strain ATCC39006.

Many enzymes use metal ions within their active sites to achieve enormous rate acceleration. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). The three-dimensional arrangement determined by X-ray crystallography provides a powerful starting point for identifying ground state interactions, but only functional studies can establish and interrogate transition state interactions. The Tetrahymena group I ribozyme is a paradigm for the study of RNA catalysis, and previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified catalytic metal ions making five contacts with the substrate atoms. Here, we have combined atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to establish transition state ligands on the ribozyme for one of the catalytic metal ions, referred to as M A . We identified the pro-S P oxygen atoms at nucleotides C208, A304, and A306 as ground state ligands for M a , verifying interactions suggested by the Azoarcus crystal structures. We further established that these interactions are present in the chemical transition state, a conclusion that requires functional studies, such as those carried out herein. Elucidating these active site connections is a crucial step toward an in-depth understanding of how specific structural features of the group I intron lead to catalysis.Phosphoryl transfer is a ubiquitous reaction in biology, yet it is extremely slow in solution (1,2). To accelerate this class of reactions to an extent compatible with life, many enzymes have evolved active sites that contain metal ions. To understand how enzymes utilize the catalytic power of metal ions, we must identify individual metal ions, define their coordination † This work was supported by a grant from the NIH (GM 49243) to D.H. and by a grant from the Howard Hughes Medical Institute to J.A.P.© 2008 American Chemical Society *Address correspondence to this author at the Department of Biochemistry, Beckman Center, B400, Stanford University. . E-mail: herschla@stanford.edu . SUPPORTING INFORMATION AVAILABLESupporting text with references to metal ion rescue experiments in protein and RNA enzymes and discussion of possible complications in TFA analysis; Table S1, providing the values of the parameters in Scheme 3 for the M B rescue of CUCG 3′S A; Table S2, providing the values of the parameters in Scheme 3 for the M C rescue of G N ; Figures S1, S2, S3, and S5, providing the data prior to normalization associated with Figure 4B,C, Figure 5B, and Figure 6B, respectively; and Figure S4, providing the comparison of M B rescue of CUCG 3′S A using two different substrates, −1r,dP and −1d,rP, for the wt and U307S P ribozymes. This material is available free of charge via the Internet at http://pubs.acs.org NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 October 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-P...