Natural and lab-evolved proteins often recognize their RNA partners with exquisite affinity. Structural analysis of such complexes can offer valuable insight into sequence-selective recognition that can be exploited to alter biological function. Here, we describe the structure of a lab-evolved RNA recognition motif (RRM) bound to the HIV-1 trans-activation response (TAR) RNA element at 1.80 Å-resolution. The complex reveals a trio of arginines in an evolved β2–β3 loop penetrating deeply into the major groove to read conserved guanines while simultaneously forming cation-π and salt-bridge contacts. The observation that the evolved RRM engages TAR within a double-stranded stem is atypical compared to most RRMs. Mutagenesis, thermodynamic analysis and molecular dynamics validate the atypical binding mode and quantify molecular contributions that support the exceptionally tight binding of the TAR-protein complex (KD,App of 2.5 ± 0.1 nM). These findings led to the hypothesis that the β2–β3 loop can function as a standalone TAR-recognition module. Indeed, short constrained peptides comprising the β2–β3 loop still bind TAR (KD,App of 1.8 ± 0.5 μM) and significantly weaken TAR-dependent transcription. Our results provide a detailed understanding of TAR molecular recognition and reveal that a lab-evolved protein can be reduced to a minimal RNA-binding peptide.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

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T he flow of genetic information in living systems is encapsulated by the central dogma of molecular biology: DNA is transcribed into RNA, which is then translated into protein. In reality, of course, the story is much more complex and interesting. But fundamentally, this is us.Our DNA is constantly changing, as a result of (among other mechanisms) spontaneous deamination, photocycloadditions involving adjacent bases (thymine dimerization), or modification of a base with a reactive chemical. While we have DNA repair machinery to revert these changes throughout our genome, some persist. Relatively minute changes in our DNA can have no obvious affect at all or can lead to new and improved function(s) in encoded RNAs or proteins. However, in some cases, changes can lead to diseases like cancer. Given the relationship between DNA sequence and disease, a holy grail for much of the last half-century has been to chemically "fix", with surgical precision, disease-relevant alterations to DNA, so-called gene editing. In addition to potential future therapeutic application, gene editing also allows researchers to reliably examine the relationship between a particular gene and disease.CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery consists of a Cas9 endonuclease and single-guide RNA (sgRNA). Once inside the nucleus of a cell, the Cas9−sgRNA complex selectively engages a DNA sequence complementary to the sgRNA, utilizing established rules for DNA−RNA base pairing. CRISPR-associated nucleases have been used for sequenceselective gene suppression by double-stranded DNA (dsDNA) cleavage and subsequent nonhomologous end joining (NHEJ). Additionally, CRISPR-associated nucleases have worked in concert with sequence-defined exogenous dsDNAs for precise gene editing through homology-directed repair (HDR). For a detailed discussion of the fascinating development of CRISPR, scholars are directed to recent perspectives. 1,2 In the context of genome engineering, CRISPR-mediated dsDNA breaks can be risky business. Additionally, many diseases are the result of single-nucleotide polymorphisms (SNPs). These mutations are more rationally (and safely) addressed using "base editing", which specifically reverts a SNP without inducing a dsDNA break and thus minimizes insertions and deletions. In seminal work, Liu and co-workers reported the first base editors, 3 which convert G•C SNPs to A•T, and established a strategy reliant on targeted base modification followed by cellular mismatch repair to fix the complementary strand.More recently, the same laboratory has developed a CRISPRbased adenosine base editing (ABE) fusion protein to "fix" C•G to T•A mutations, which account for approximately half of all known pathogenic SNPs. In this system, a catalytically impaired Cas9 protein is fused to a deoxyadenosine deaminase enzyme.

Drug-resistant tuberculosis infections are a major challenge to global public health. Much effort has been invested in identifying new drug targets in the causative pathogen, Mycobacterium tuberculosis .