Spotting off-targets from gene editing Unintended genomic modifications limit the potential therapeutic use of gene-editing tools. Available methods to find generally do not work in vivo or detect single-nucleotide changes. Three papers this issue report new for monitoring tools (see Perspective by Kempton and Qi). Wienert et al. followed recruitment a DNA repair protein breaks induced CRISPR-Cas9, enabling unbiased detection off-target cellular animal models. Zuo identified without...

Abstract Repair of double strand DNA breaks (DSBs) can result in gene disruption or modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away other outcomes. Here, we utilize a pooled CRISPR screen define host cell involvement between Cas9 DSB plasmid stranded (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor genes act repress HDR. Small molecule inhibition one these...

Abstract The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, activity these Cas9–sgRNA combinations varies widely at different loci and in cell types. Thus, disrupting genes polyploid lines or when using poorly performing sgRNAs require extensive downstream screening identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption...

Abstract CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce have been devised, all imperfect. Here, we report that can be shielded from the active Cas9•single guide RNA (sgRNA) complex through co-administration of dead-RNAs (dRNAs), truncated RNAs direct Cas9 binding not cleavage. dRNAs effectively suppress wide-range off-targets with minimal optimization while...

Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced import proteins into peroxisomes. RNF146-mediated loss depended on stabilization and activity poly(ADP-ribose)...

DNA double-stranded breaks (DSBs) are especially toxic events that can be reversed by homology-directed repair (HDR), wherein information is copied from an intact template molecule. RAD51 mediates initial DSB/template pairing during homology search. A major challenge in understanding search cells the lack of tools to monitor this process. We developed proximity identification sequencing (RaPID-seq), a sensitive method marks all candidate templates searched RAD51. find HDR hierarchical, such...

Abstract The FANCD2-FANCI heterodimer contributes to DNA repair at interstrand crosslinks and sites of replication stress. This complex has been physically mechanistically linked double-strand break (DSB) repair, but its role in that process remains undefined. Here we show the dynamically interacts with open chromatin regions, including transient, DSB-induced chromatin, where it can be stabilized by co-activation kinase ATM Fanconi anemia core ubiquitin ligase. loaded stabilizes promotes...

We describe a strategy to boost the efficiency of gene editing via homology-directed repair (HDR) by covalently modifying template DNA with interstrand crosslinks. Crosslinked templates (xHDRTs) increase Cas9-mediated efficiencies up fivefold in K562, HEK293T, U2OS, iPS and primary T cells. Increased from xHDRTs is driven events on molecule requires ataxia telangiectasia Rad3-related (ATR) kinase components Fanconi anemia pathway.

Eukaryotic cells must inhibit re-initiation of DNA replication at each the thousands origins in their genome because can generate genomic alterations with extraordinary frequency. To minimize probability from so many origins, use a battery regulatory mechanisms that reduce activity initiation proteins. Given global nature these mechanisms, it has been presumed all are inhibited identically. However, re-initiate diverse efficiencies when disabled, and this diversity cannot be explained by...

The heterohexameric ATPases associated with diverse cellular activities (AAA)-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses energy from ATP hydrolysis mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited binding motor's N-terminal domains or N terminally bound cofactors. Here, we use structural biochemical techniques...

Abstract Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability be genetically encoded produce magnetic resonance signals by increasing the rate of diffusion cells. However, concerns regarding effects Aqp1 overexpression increased membrane diffusivity on cell physiology have limited widespread use as deep-tissue reporter. In this study, we present evidence that generates strong diffusion-based without...

Abstract Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced import proteins into peroxisomes. RNF146-mediated loss depended on stabilization and activity...

Eukaryotic cells are constantly adapting to their environment by remodeling gene expression, proteome, and morphology. One fascinating example of cellular adaptation versatility is the peroxisome, a membrane-bound organelle that remodeled across evolution development meet specific metabolic needs cells. Study peroxisome has trailed other organelles such as endoplasmic reticulum or mitochondrion, perhaps because peroxisomes lack single essential function. However, actually why it so important...

Eukaryotic cells repair DNA double strand breaks (DSBs) using two strategies: end joining (EJ), which ligates the ends of lesion back together, and homologous recombination (HR), uses a template molecule to replace sequence spanning DSB. Previous work by our lab others established that Fanconi Anemia (FA) pathway, especially FANCD2, binds DSBs regulates DSB outcomes. However, precise role played FANCD2 in remained undefined. Here we report localizes both during this localization depends on...

The heterohexameric AAA-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses energy from ATP hydrolysis mechanically thread substrate proteins through its central pore, thereby unfolding them. In related motors, substrates are recruited binding motor's N-terminal domains or N-terminally bound co-factors. Here we use structural biochemical techniques characterize function N1 domain in Pex6 budding yeast, S. cerevisiae....

Abstract The PEX1/PEX6 AAA-ATPase is required for the biogenesis and maintenance of peroxisomes. Mutations in HsPEX1 HsPEX6 disrupt peroxisomal matrix protein import are leading cause Peroxisome Biogenesis Disorders (PBDs). most common disease-causing mutation PEX1 Hs G843D allele, which results a reduction import. Here we demonstrate that vitro homologous yeast mutant, Sc Pex1 G700D , reduces stability Pex1’s active D2 ATPase domain impairs assembly with Pex6, but can still form an motor....

Repair of double strand DNA breaks (DSBs) can result in gene disruption or precise modification via homology directed repair (HDR) from a templating donor DNA. During genome editing, altering cellular responses to DSBs may be an effective strategy rebalance editing outcomes towards HDR and away other pathways. To identify factors that regulate double-stranded (dsDonor), we utilized pooled screen define the consequences thousands individual knockdowns during Cas9-initiated plasmid donor. We...

Abstract Cas9 endonuclease can be targeted to genomic sequences by varying the sequence of single guide RNA (sgRNA). The activity these Cas9-sgRNA combinations varies widely at different loci and in cell types. Thus, disrupting genes polyploid lines, or using inefficient sgRNAs, require extensive downstream screening identify homozygous clones. We have found that linear, non-homologous oligonucleotide DNA greatly stimulates Cas9-mediated gene disruption absence homology-directed repair. This...

Abstract Co-introduction of targeted nucleases and DNA/RNA templates encoding new genomic sequence is the basis for rapid, effective, iterable gene editing workflows therapeutic, agricultural, basic science applications. Extensive optimization reagent delivery nuclease activity have improved genome workflows, but comparatively few efforts been made to alter template molecules. Here, we report DNA modified with interstrand crosslinks (ICLs) – xHDRTs - increases frequencies in Cas9-directed by...

The heterohexameric AAA-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses energy from ATP hydrolysis mechanically thread substrate proteins through its central pore, thereby unfolding them. In related motors, substrates are recruited binding motor's N-terminal domains or N-terminally bound co-factors. Here we use structural biochemical techniques characterize function N1 domain in Pex6 budding yeast,