Tripartite motif (TRIM) proteins make up a large family of coiledcoil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the Nterminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication.antiparallel dimer | disulfide crosslinking | X-ray crystallography T ripartite motif (TRIM) proteins make up the largest superfamily of RING E3 ubiquitin ligases, with more than 100 members in the human proteome (1, 2). TRIM proteins function in a variety of cellular pathways, and many regulate innate immunity and/or mediate antiviral responses. Antiviral TRIMs include TRIM25, which regulates the IFN response to RNA viruses (3, 4), and TRIM5α, which senses and inhibits early stages of retroviral replication (5, 6).TRIM proteins share a common N-terminal domain organization, termed the tripartite or RBCC (RING, B-box, coiled-coil) motif, followed by variable C-terminal protein recognition domains ( Fig. 1 A and B). "Linker" segments of unknown structure typically separate both the RING and B-box domains (L1) and the coiledcoil and terminal effector domains (L2). The coiled-coil region mediates oligomerization, and both homooligomeric and heterooligomeric TRIMs have been described (7-13). Furthermore, many TRIM proteins form higher-order assemblies in vitro and form punctate or fibrous structures in cells (14-16). For example, TRIM5α assembly allows the protein to function as a cytosolic patternrecognition receptor that can intercept the incoming capsids of diverse retroviruses, including HIV-1 (6). This results in species-specific "restriction" of viral replication (5), capsid dissociation (5, 6), and induction of innate immune responses (17). Retroviral capsids are recognized through a remarkable mechanism of multivalent pattern recognition. TRIM5α forms a homodimer (10, 11, 18), which can further assemble into a 2D lattice of linked hexagons (18). The hexagonal TRIM5α net matches the symmetry and spacing of the retroviral capsid surface lattice, thereby positioning multiple C-terminal B30.2/SPRY domains to interact with their repeating binding epitopes on the capsid.Struct...

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

Transcriptional adaptation is a recently described phenomenon by which a mutation in one gene leads to the transcriptional modulation of related genes, termed adapting genes. At the molecular level, it has been proposed that the mutant mRNA, rather than the loss of protein function, activates this response. While several examples of transcriptional adaptation have been reported in zebrafish embryos and in mouse cell lines, it is not known whether this phenomenon is observed across metazoans. Here we report transcriptional adaptation in C. elegans, and find that this process requires factors involved in mutant mRNA decay, as in zebrafish and mouse. We further uncover a requirement for Argonaute proteins and Dicer, factors involved in small RNA maturation and transport into the nucleus. Altogether, these results provide evidence for transcriptional adaptation in C. elegans, a powerful model to further investigate underlying molecular mechanisms.

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

22 23Wierson, Welker, Almeida et al. GeneWeld: a method for efficient targeted integration directed by short homology 2 1 Wierson et al. describe a targeted integration strategy, called GeneWeld, and a vector 2 series for gene tagging, pGTag, which promote highly efficient and precise targeted integration 3 in zebrafish, pig fibroblasts, and human cells. This approach establishes an effective genome 4 engineering solution that is suitable for creating knock-in mutations for functional genomics and 5 gene therapy applications. The authors describe high rates of germline transmission (50%) for 6 targeted knock-ins at eight different zebrafish loci and efficient integration at safe harbor loci in 7 porcine and human cells. Abstract 11Choices for genome engineering and integration involve high efficiency with little or no 12 target specificity or high specificity with low activity. Here, we describe a targeted integration 13 strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene 14Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, 15 pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that 16 in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing 17 as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the 18 homology arms are exposed with a double strand break in vivo. Our results suggest that the 19 length of homology is not important in the design of knock-in vectors but rather how the 20 homology is presented to a double strand break in the genome. Given our results targeting 21 multiple loci in different species, we expect the accompanying protocols, vectors, and web 22 interface for homology arm design to help streamline gene targeting and applications in 23 CRISPR and TALEN compatible systems. 25Keywords