Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of noncanonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically ...produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (k
/K
) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing noncanonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.
The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently ...convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.
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The spontaneous deamination of cytosine is a major source of transitions from CG to TA base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently ...convert targeted AT base pairs to GC could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of AT to GC in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPRCas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted AT base pairs efficiently to GC (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.
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IJS, KISLJ, NUK, SBMB, UL, UM, UPUK
Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion using noncanonical amino acids, yet its structure and function are not completely understood. Here we describe the crystal ...structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNA
. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNA
recognition by PylRS is anticodon independent: the anticodon does not contact the enzyme. Then, using standard microbiological culture equipment, we established a new method for laboratory evolution-a noncontinuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. Finally, we employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a noncanonical amino acid.
A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human ...muscular dystrophies. Here, we show that one of these mutations, sapje , disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy ( DMD ) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases.
Th17 cells play a critical role in the adaptive immune response against extracellular bacteria, and the possible mechanisms by which they can protect against infection are of particular interest. In ...this study, we describe, to our knowledge, a novel IL-1β dependent pathway for secretion of the antimicrobial peptide IL-26 from human Th17 cells that is independent of and more rapid than classical TCR activation. We find that IL-26 is secreted 3 hours after treating PBMCs with
as compared with 48 hours for IFN-γ and IL-17A. IL-1β was required for microbial ligand induction of IL-26 and was sufficient to stimulate IL-26 release from Th17 cells. Only IL-1RI
Th17 cells responded to IL-1β, inducing an NF-κB-regulated transcriptome. Finally, supernatants from IL-1β-treated memory T cells killed
in an IL-26-dependent manner. These results identify a mechanism by which human IL-1RI
"antimicrobial Th17 cells" can be rapidly activated by IL-1β as part of the innate immune response to produce IL-26 to kill extracellular bacteria.
Rational design of RNA ligands continues to be a formidable challenge, but the potential powerful applications in biology and medicine catapults it to the forefront of chemical research. Indeed, ...small molecule and macromolecular intervention are attractive approaches, but selectivity and cell permeability can be a hurdle. An alternative strategy is to use molecules of intermediate molecular weight that possess large enough surface area to maximize interaction with the RNA structure but are small enough to be cell-permeable. Herein, we report the discovery of nontoxic and cell-permeable branched peptide (BP) ligands that bind to TAR RNA in the low micromolar range from on-bead high-throughput screening of 4,096 compounds. TAR is a short RNA motif in the 5′-UTR of HIV-1 that is responsible for efficient generation of full RNA transcripts. We demonstrate that BPs are selective for the native TAR RNA structure and that “branching” in peptides provides multivalent interaction, which increases binding affinity to RNA.
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We report branched peptide boronic acids (BPBAs) that bind to RRE IIB from an on-bead high-throughput screening of a 3.3.4-library (46 656 compounds). We demonstrate that boronic acids are tunable ...moieties that afford a novel binding mode towards RNA.
In this Article, owing to an error during the production process, in Fig. 1a, the dark blue and light blue wedges were incorrectly labelled as 'G•C → T•A' and 'G•C → A•T', instead of 'C•G → T•A' and ...'C•G → A•T', respectively. Fig. 1 has been corrected online.
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