Precise Deletion Research Articles

Abstract 4802: Functional analysis of cancer associated mitochondrial genome alterations

Abstract It is well documented that a variety of alterations can occur in the mitochondrial genome during human tumor development. These alterations often correlate with cancer phenotypes like treatment resistance and metastasis. Alterations to the mitochondrial genome may therefore be a biomarker for early detection, prognosis and/or predictor of therapeutic sensitivity. However, a method to easily and quickly create isogenic populations of human cells with mitochondrial genome specific alterations is not available. To combat this, we have developed a technology using transient expression of mitochondria targeted TAL domain nickases and TAL endonucleases (TALENs). This the first reliable method to introduce specific mutations into the mitochondrial genome of mammalian cells. We have efficiently induced mtDNA deletions in zebrafish embryos, human and mouse cell lines. In mouse NIH3T3 fibroblasts, we deleted a mitochondrial genome region corresponding to the same disease sequence seen in the human Kearns-Sayre Syndrome (KSS). The deletion resulted in a measurable phenotype including decreased mitochondria basal respiration, ATP production and maximal respiration. We are expanding this work by engineering targeted and precise mtDNA deletions in conjunction with known cancer initiating nuclear mutations in mammalian cells, as well as systemically in mice. These tools will allow us to determine the effect mitochondrial alterations have on the transformation of cancer cells harboring known cancer causing nuclear mutations. We are also developing an inducible system for expressing mitoTALENs, making it possible to create “conditional” loss of function mutations in mitochondrial genes in human cell lines, including induced pluripotent stem cells (iPSCs). This inducible model system will be highly useful in settings where the mitochondrial deletion may cause a selective disadvantage during early cellular differentiation. Citation Format: Neil Otto. Functional analysis of cancer associated mitochondrial genome alterations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4802.

Evidence that miR-152-3p is a positive regulator of SETDB1-mediated H3K9 histone methylation and serves as a toggle between histone and DNA methylation

SETDB1 is a histone methyltransferase that converts H3K9me2 to H3K9me3. SETDB1 activity and H3K9me3 are crucial for the formation of obligately silenced heterochromatin such as that of centromeres. Here we show that a microRNA, miR-152-3p, is involved in the regulation of SETDB1 protein levels, but surprisingly, miR-152-3p plays a positive regulatory role for SETDB1 expression. Inhibition of miR-152-3p by anti-miR treatment resulted in a robust reduction in SETDB1 protein levels, though SETDB1 mRNA levels were unaffected. This was also accompanied by a blockade of the biochemical pathway proceeding from H3K9me2 to H3K9me3 as evidenced by quantitative nucleosome ELISA assays that showed that H3K9me2 accumulates in cells treated with an anti-miR that targets miR-152-3p. In addition, the action of a miR-152-3p mimic increased flux of the reaction leading to H3K9me3. We also performed site-directed mutagenesis of three predicted miR-152-3p target recognition sequences to yield three precise deletions. Deletion of one of the three sites recapitulated the positive regulatory aspect of the action of miR-152-3p upon SETDB1 expression in a luciferase reporter assay. Previous studies have shown that miR-152-3p negatively regulates DNMT1, the sole maintenance DNA methyltransferase which is required for levels of 5-methylcytosine levels within DNA. Our results shown that miR-152-3p positively regulates the production of H3K9me3 by regulating the production of SETDB1. Therefore, our findings provide strong evidence that miR-152-3p can serve as a toggle switch that regulates the balance between DNA methylation and H3K9 histone methylation in constitutive heterochromatin.

Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals

Gene-editing technology can artificially modify genetic material of targeted loci by precise insertion, deletion, or replacement in the genomic DNA. In recent years, with the developments of zinc-finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) technologies, such precise modifications of the animal genomes have become possible. Although gene-editing tools, such as CRISPR/Cas9, can efficiently generate double-strand breaks (DSBs) in mammalian cells, the homology-directed repair (HDR) mediated knock-in (KI) efficiency is extremely low. In this review, we briefly describe the current development of gene-editing tools and summarize the recent strategies to enhance the CRISPR/Cas9- mediated KI efficiency, which will provide a reference for the generation of human disease models, research on gene therapy and livestock genetic improvement.

Engineering of targeted megabase-scale deletions in human induced pluripotent stem cells

Recurrent chromosomal deletions spanning several megabases are often found in hematological malignancies. The ability to engineer deletions in model systems to functionally study their effects on the phenotype would enable, first, determination of whether a given deletion is pathogenic or neutral and, second, identification of the critical genes. Incomplete synteny makes modeling of deletions of megabase scale challenging or impossible in the mouse or other model organisms. Furthermore, despite the breakthroughs in targeted nuclease technologies in recent years, engineering of megabase-scale deletions remains challenging and has not been achieved in normal diploid human cells. Large deletions of the long arm of chromosome 7 (chr7q) occur frequently in myelodysplastic syndrome (MDS) and are associated with poor prognosis. We previously found that we can model chr7q deletions in human induced pluripotent stem cells (iPSCs) using a modified Cre-loxP strategy. However, this strategy did not afford control over the length and boundaries of the engineered deletions, which were initiated through random chromosome breaks. Here we developed strategies enabling the generation of defined and precise chromosomal deletions of up to 22 Mb, using two different strategies: “classic” Cre-loxP recombination and CRISPR/Cas9-mediated DNA cleavage. As proof of principle, we illustrate that phenotypic characterization of the hematopoiesis derived from these iPSCs upon in vitro differentiation allows further definition of the critical region of chr7q whose hemizygosity impairs hematopoietic differentiation potential. The strategies we present here can be broadly applicable to engineering of diverse chromosomal deletions in human cells.

Efficient CRISPR-Cas9-Mediated Gene Ablation in Human Keratinocytes to Recapitulate Genodermatoses: Modeling of Netherton Syndrome

Current efforts to find specific genodermatoses treatments and define precise pathogenesis mechanisms require appropriate surrogate models with human cells. Although transgenic and gene knockout mouse models for several of these disorders exist, they often fail to faithfully replicate the clinical and histopathological features of the human skin condition. We have established a highly efficient method for precise deletion of critical gene sequences in primary human keratinocytes, based on CRISPR-Cas9-mediated gene editing. Using this methodology, in the present study we generated a model of Netherton syndrome by disruption of SPINK5. Gene-edited cells showed absence of LEKTI expression and were able to recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of Netherton syndrome, after grafting to immunodeficient mice and in organotypic cultures. To validate the model as a platform for therapeutic intervention, we tested an ex vivo gene therapy approach using a lentiviral vector expressing SPINK5. Re-expression of SPINK5 in an immortalized clone of SPINK5-knockout keratinocytes was capable of reverting from Netherton syndrome to a normal skin phenotype in vivo and in vitro. Our results demonstrate the feasibility of modeling genodermatoses, such as Netherton syndrome, by efficiently disrupting the causative gene to better understand its pathogenesis and to develop novel therapeutic approaches.

Acquiring Resistance Against a Retroviral Infection via CRISPR/Cas9 Targeted Genome Editing in a Commercial Chicken Line.

Genome editing technology provides new possibilities for animal breeding and aid in understanding host-pathogen interactions. In poultry, retroviruses display one of the most difficult pathogens to control by conventional strategies such as vaccinations. Avian leukosis virus subgroup J (ALV-J) is an oncogenic, immunosuppressive retrovirus that causes myeloid leukosis and other tumors in chickens. Severe economic losses caused by ALV-J remain an unsolved problem in many parts of the world due to inefficient eradication strategies and lack of effective vaccines. ALV-J attachment and entry are mediated through the specific receptor, chicken Na+/H+ exchanger type 1 (chNHE1). The non-conserved amino acid tryptophan 38 (W38) in chNHE1 is crucial for virus entry, making it a favorable target for the introduction of disease resistance. In this study, we obtained ALV-J-resistance in a commercial chicken line by precise deletion of chNHE1 W38, utilizing the CRISPR/Cas9-system in combination with homology directed repair. The genetic modification completely protected cells from infection with a subgroup J retrovirus. W38 deletion did neither have a negative effect on the development nor on the general health condition of the gene edited chickens. Overall, the generation of ALV-J-resistant birds by precise gene editing demonstrates the immense potential of this approach as an alternative disease control strategy in poultry.

Investigation into the Role of Histone H3 Lysine 4 in Developmental Gene Regulation

Histone H3 lysine 4 (H3K4) methylation is an evolutionarily‐conserved modification of chromatin closely linked to gene activation. H3K4 monomethylation (me1) is associated with active enhancers and flanks promoters. H3K4 dimethylation (me2) is enriched at tissue‐specific transcription factor binding sites, enhancers, and promoter edges. H3K4 trimethylation (me3) defines active transcriptional initiation at promoters. H3K4me1, me2, and me3 in Drosophila melanogaster is mediated by the methyltransferases Trr, dSet1, and Trx. The human homologs of these enzymes (MLL3/4, SET1A/B, and MLL1/2, respectively) perform conserved roles in human gene regulation and are commonly mutated in cancers. While these histone‐modifying enzymes have been found to be major regulators of development, differentiation, and disease, their enzymatic activity is not limited to histones and they also have non‐enzymatic functions. For these reasons, the specific role of H3K4 modification in transcriptional regulation is not understood. To directly test the function of H3K4 in animal development, we mutated H3K4 in Drosophila melanogaster. In this approach, the endogenous wild‐type histone genes are replaced with transgenic versions that have been mutated to encode a non‐modifiable residue (H3K4R). Notably, Drosophila is the only animal experimental system in which histone gene replacement can be performed because histone genes exist in high copy numbers and at multiple locations throughout the genome of most animals. By contrast, the canonical histone genes in Drosophila are clustered in a single chromosomal locus, allowing for its precise deletion (1). Previously, we showed that wild‐type replacement transgene supports the propagation of a stable stock lacking all endogenous histone genes (1). By contrast, here we find that H3K4R replacement transgenes result in embryonic lethality. Thus, H3K4 modification is essential for animal development. In future work, we will determine the effects of H3K4 mutation on gene expression through RNA‐seq analysis, as well as its impact on the histone post‐translational modification landscape through mass spectrometry. Together, this work will extend our understanding of the mechanistic role that histone modifications contribute to developmental gene regulation.Support or Funding InformationNIH to DJM R35‐GM128851

The C2C-MCT Domain of Munc13 is Essential for Priming Synaptic Vesicles

Munc13 and Complexin (CPX) play pivotal roles in both spontaneous and calcium triggered synaptic vesicle (SV) fusion. Using a high-throughput behavioral assay on chemically mutagenized C. elegans lacking CPX, we identified an early-stop mutation at the beginning of the C2C domain near the C-terminal end of the Munc13 ortholog, UNC-13. Munc13 C2C is implicated in tethering SVs to the plasma membrane. However, its mechanism of tethering vesicles is not well understood. Also, to our knowledge, the C2C domain of Munc13 has not been isolated and characterized in any system to date. Here, we report that one or two alpha helices of MUN domain preceding C2C and the predicted alpha helices of the last 60 residues (MCT) are crucial for C2C folding and stability. The precise deletion of C2C or MCT impairs synaptic transmission, decreases SV priming, and disrupts nervous system function in C. elegans. To understand the role of C2C domain in tethering and priming SVs, we characterized the membrane binding properties of Munc13 C2C-MCT using various approaches including co-sedimentation, co-flotation and tryptophan fluorescence. We found that the C2C-MCT domain binds membranes in a calcium- and charge-independent manner. Using behavioral assays in worms, a single point mutation in a predicted loop of C2C domain impaired nervous system function but did not affect lipid binding. In contrast, disrupting a hydrophobic patch in MCT abrogated lipid binding but did not affect nervous system function. Finally, artificially tethering the UNC-13 MUN domain to SVs rescued the unc-13 null mutant while plasma membrane tethered UNC-13 failed to restore function. On basis of these results, we conclude that SV localization of C2C-MCT is critical for efficient priming by Munc13, relies on protein-protein interactions, and acts upstream of the clamping function of CPX.

Bombardier Enables Delivery of Short-Form Bomanins in the Drosophila Toll Response.

Toll mediates a robust and effective innate immune response across vertebrates and invertebrates. In Drosophila melanogaster, activation of Toll by systemic infection drives the accumulation of a rich repertoire of immune effectors in hemolymph, including the recently characterized Bomanins, as well as the classical antimicrobial peptides (AMPs). Here we report the functional characterization of a Toll-induced hemolymph protein encoded by the bombardier (CG18067) gene. Using the CRISPR/Cas9 system to generate a precise deletion of the bombardier transcriptional unit, we found that Bombardier is required for Toll-mediated defense against fungi and Gram-positive bacteria. Assaying cell-free hemolymph, we found that the Bomanin-dependent candidacidal activity is also dependent on Bombardier, but is independent of the antifungal AMPs Drosomycin and Metchnikowin. Using mass spectrometry, we demonstrated that deletion of bombardier results in the specific absence of short-form Bomanins from hemolymph. In addition, flies lacking Bombardier exhibited a defect in pathogen tolerance that we trace to an aberrant condition triggered by Toll activation. These results lead us to a model in which the presence of Bombardier in wild-type flies enables the proper folding, secretion, or intermolecular associations of short-form Bomanins, and the absence of Bombardier disrupts one or more of these steps, resulting in defects in both immune resistance and tolerance.

A novel gross deletion and breakpoint junction sequence analysis of ATP7B in a Chinese family with Wilson disease using next‑generation sequencing and Sanger sequencing.

Wilson disease (WD) is a rare autosomal recessive genetic disorder that causes abnormal copper metabolism, resulting in pathological accumulation of copper in the liver, brain and other organs. Mutations in the ATPase copper transporter 7B (ATP7B) gene, which encodes a membrane P-type adenosine triphosphatase, have been identified as being responsible for WD. The present study analyzed clinical data and collected DNA samples from a pediatric patient with WD and her healthy parents. Mutation screening for ATP7B was performed using direct sequencing, multiplex ligation-dependent probe amplification(MLPA), next-generation sequencing (NGS) and Sanger sequencing of the breakpoint junction sequence. The patient (age, 2.7 years) presented with early-onset hepatic disease. The present study identified compound heterozygous mutations of ATP7B, including a heterozygous mutation (p.Arg1,041Trp) and a novel heterozygous gross deletion of a 57,771 bp fragment (chr13: 52490972-52548742) (GRCh37) from partial exon2- exon21 to external ATP7B sequence (15.833bp) in the patient. Analysis of the family members of the patient showed that the missense mutation and the gross deletion mutation were inherited from her mother and father, respectively. Microhomology and inverted repeat sequences, which may mediate the deletion mutation, were identified through sequence analysis on both sides of the breakpoints of this deletion. The present study provided additional information on the genotypic spectrum of the ATP7B gene, particularly with regard to early onset hepatic disease, as observed in the present patient with WD. The identification of the precise breakpoint junction sequence warrants further investigation of DNA break and recombination mechanisms. In detecting precise deletions, the NGS associated with Sanger sequencing of breakpoint junction sequence have been found to have more advantages than MLPA.

Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations

The functional effect of a gene edit by designer nucleases depends on the DNA repair outcome at the targeted locus. While non-homologous end joining (NHEJ) repair results in various mutations, microhomology-mediated end joining (MMEJ) creates precise deletions based on the alignment of flanking microhomologies (µHs). Recently, the sequence context surrounding nuclease-induced double strand breaks (DSBs) has been shown to predict repair outcomes, for which µH plays an important role. Here, we survey naturally occurring human deletion variants and identify that 11 million or 57% are flanked by µHs, covering 88% of protein-coding genes. These biologically relevant mutations are candidates for precise creation in a template-free manner by MMEJ repair. Using CRISPR-Cas9 in human induced pluripotent stem cells (hiPSCs), we efficiently create pathogenic deletion mutations for demonstrable disease models with both gain- and loss-of-function phenotypes. We anticipate this dataset and gene editing strategy to enable functional genetic studies and drug screening.

Native display of a huge homotrimeric protein fiber on the cell surface after precise domain deletion

AtaA, a trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, exhibits nonspecific, high adhesiveness to abiotic surfaces. For identification of the functional domains of AtaA, precise design of domain-deletion mutants is necessary so as not to cause undesirable structural distortion. Here, we designed and constructed three types of AtaA mutants from which the same domain, FGG1, was deleted. The first mutant was designed to preserve the periodicity of hydrophobic residues in the coiled-coil segments sandwiching the deleted region. After the deletion, the protein was properly displayed on the cell surface and had the same adhesive function as the wild type. Transmission electron microscopy (TEM) imaging and circular dichroism (CD) spectroscopy showed that its isolated passenger domain had the same fiber structure as in the AtaA wild type. In contrast, a mutant designed to disturb the coiled-coil periodicity at the deletion site failed to reach the cell surface. Although secretion occurred for the mutant designed with a flexible connector between the coiled coils, the cells exhibited a decrease in adhesiveness. Furthermore, TEM imaging of the mutant fibers showed bending at the fiber tip and changes in their CD spectrum indicated a decrease in secondary structure content. Thus, we succeeded to natively display the huge homotrimeric fiber structure of AtaA on the cell surface after precise deletion of a domain, maintaining the proper folding state and adhesive function by preserving its coiled-coil periodicity. This strategy enables us to construct various domain-deletion mutants of AtaA without structural distortion for complete functional mapping.

Generation of deletions and precise point mutations in Dictyostelium discoideum using the CRISPR nickase.

The CRISPR/Cas9 system enables targeted genome modifications across a range of eukaryotes. Although we have reported that transient introduction of all-in-one vectors that express both Cas9 and sgRNAs can efficiently induce multiple gene knockouts in Dictyostelium discoideum, concerns remain about off-target effects and false-positive amplification during mutation detection via PCR. To minimise these effects, we modified the system to permit gene deletions of greater than 1 kb via use of paired sgRNAs and Cas9 nickase. An all-in-one vector expressing the Cas9 nickase and sgRNAs was transiently introduced into D. discoideum, and the resulting mutants showed long deletions with a relatively high efficiency of 10–30%. By further improving the vector, a new dual sgRNA expression vector was also constructed to allow simultaneous insertion of two sgRNAs via one-step cloning. By applying this system, precise point mutations and genomic deletions were generated in the target locus via simultaneous introduction of the vector and a single-stranded oligonucleotide template without integrating a drug resistance cassette. These systems enable simple and straightforward genome editing that requires high specificity, and they can serve as an alternative to the conventional homologous recombination-based gene disruption method in D. discoideum.

Abstract 4782: STACT: A novel tumor-targeting, systemically-administered delivery platform capable of targeting intractable pathways and precise immunomodulation of the tumor microenvironment

Abstract We have engineered a tumor-targeted, microbial immunotherapy platform called STACT (Salmonella Typhimurium (Attenuated) and Checkpoint Therapy). The platform strain has been engineered to increase tumor-specific enrichment, reduce immunosuppressive inflammation and enhance tolerability. Upon phagocytosis by tumor-resident immune cells, the microbe delivers plasmid DNA, which can either encode inhibitory microRNAs to knockdown immune targets of interest, or encode immuno-modulatory cDNA expression cassettes, alone or in specific combinations. The STACT platform strain is over 10,000-fold attenuated for virulence due to disruptions in the msbB and purI genes, that result in the detoxification of the surface LPS and purine/adenosine auxotrophy, respectively. The STACT platform strain required addition of exogenous adenosine or purines at concentrations found in the tumor micro-environment, but not in healthy tissue for replication in vitro. The STACT platform strain was unable to replicate significantly in infected macrophage cell lines, but was able to colonize tumors in mice and deliver functional plasmids. This strain was further engineered by precise genome deletions of the fljB and fliC genes, encoding the bacterial flagellar subunits that are strong TLR5 agonists and induce inflammasome-mediated pyroptosis in macrophages. Deletion of the flagellin genes prevented bacterial cell motility but did not affect tumor-specific colonization after IV administration, and tumor enrichment was observed at levels over 100,000 times greater than in spleens. A plasmid maintenance system was engineered by deletion of the chromosomal asd gene and complementation of the asd gene on a copy-number optimized plasmid to ensure plasmid maintenance in vivo. The asd system allowed for significantly improved plasmid retention in vivo without antibiotic selection. Furthermore, the vector incorporates immunostimulatory CpG motifs into the strain to help promote a TLR9-mediated adaptive immune response to tumor antigens. A library of inhibitory RNAi’s against a set of immuno-modulatory targets, including TREX1, PD-L1, and TGF-beta were screened for optimal knockdown of gene expression by qPCR and western blot. Pairwise combinatorial knockdown of specific targets was also observed in human cells. IV administration of STACT encoding TGF-beta RNAi demonstrated significant tumor growth inhibition in a subcutaneous tumor model. STACT is a highly attenuated microbial immunotherapy platform engineered to deliver immunomodulatory molecules to phagocytic cells in the tumor microenvironment after systemic administration. This platform can be engineered to knock down combinations of immune checkpoints or express immunostimulatory genes in a single therapeutic modality. Citation Format: Chris S. Rae, Marina Besprozvannaya, John Faulhaber, Anastasia M. Makarova, Beverly King, Laura Hix Glickman, Christopher D. Thanos, Justin Skoble. STACT: A novel tumor-targeting, systemically-administered delivery platform capable of targeting intractable pathways and precise immunomodulation of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4782.

Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination

CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms, but its use in pigs to generate precise genetic modifications has seldom been described. Myostatin (MSTN) is a well-characterized negative regulator of muscle development, and natural mutations in this gene cause a double-muscled phenotype in many species. However, to the best of our knowledge, no naturally occurring mutation in MSTN has been found in pigs. In addition, no living pig models with sophisticated modifications orthologous to natural mutations in MSTN have yet been reported. In this study, we exploited the CRISPR/Cpf1 system to introduce a predefined modification orthologous to the natural MSTN mutation found in Belgian Blue cattle (thus known as the Belgian Blue mutation). Our research demonstrated that the cutting efficiency of CRISPR/Cpf1 was 12.3% in mixed porcine fetal fibroblasts in drug free medium, and 41.7% in clonal colonies obtained using G418 selection. Then, the Cpf1-sgRNA vector, ssODN template, and a self-excision cassette were co-transfected into porcine fetal fibroblasts. After G418 selection, 8 clonal colonies were examined and 5 with genetic modification were found. Of these 5, 2 harbored the precise 11-bp deletion. Using 1 heterozygous clonal colony, 2 cloned Duroc piglets were successfully generated, which was heterozygous for the Belgian Blue mutation. In summary, our results demonstrate that CRISPR/Cpf1 system can be used efficiently to generate double-stranded breaks, and also to mediate homologous recombination to introduce precise genomic modifications in pigs.

Mycobacteriophage ZoeJ: A broad host-range close relative of mycobacteriophage TM4

A collection of over 1600 sequenced bacteriophages isolated on a single host strain, Mycobacterium smegmatis mc2155, can be grouped into over two dozen types that have little or no nucleotide sequence similarity to each other. One group, Cluster K, can be divided into several subclusters, and the well-characterized and much exploited phage TM4 lies in Subcluster K2. Many of the Cluster K phages have broad host ranges and infect both fast- and slow-growing mycobacterial strains. Here we describe phage ZoeJ, a new Subcluster K2 member, which infects a broad spectrum of mycobacterial hosts including M. smegmatis, Mycobacterium tuberculosis, and Mycobacterium avium. ZoeJ has extensive sequence similarity to TM4, and comparative analysis reveals the precise deletion conferring the lytic phenotype of TM4. The ZoeJ immunity repressor was identified as gene 45, which is prophage-expressed, is required for lysogeny, and is sufficient to confer superinfection immunity to ZoeJ. ZoeJ gp45 also confers immunity to Subcluster K2 phage Milly, and Subcluster K1 phages Adephagia and CrimD, but surprisingly not to TM4. RNAseq analysis reveals the temporal pattern of early and late gene expressions in ZoeJ lytic growth and suggests a role for the ESAS motifs for gene regulation.

Genomic Sequence of the Prophage-Free Lactococcus lactis Strain IL6288.

We report here the genome sequence of IL6288, a prophage-free derivative of Lactococcus lactis subsp. lactis strain IL1403, and confirm precise deletion of all prophages. Several single-nucleotide variations and an extra copy of the IS981 element, apparently having a minor influence on cell physiology, were also detected in the IL6288 genome.

Orthogonal Cas9\u2013Cas9 chimeras provide a versatile platform for genome editing

The development of robust, versatile and accurate toolsets is critical to facilitate therapeutic genome editing applications. Here we establish RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, as versatile genome engineering systems. In both of these formats, Cas9-Cas9 fusions display an expanded targeting repertoire and achieve highly specific genome editing. Dual-nuclease Cas9-Cas9 chimeras have distinct advantages over monomeric Cas9s including higher target site activity and the generation of predictable precise deletion products between their target sites. At a therapeutically relevant site within the BCL11A erythroid enhancer, Cas9-Cas9 nucleases produced precise deletions that comprised up to 97% of all sequence alterations. Thus Cas9-Cas9 chimeras represent an important tool that could be particularly valuable for therapeutic genome editing applications where a precise cleavage position and defined sequence end products are desirable.

True Homoplasy of Retrotransposon Insertions in Primates.

How reliable are the presence/absence insertion patterns of the supposedly homoplasy-free retrotransposons, which were randomly inserted in the quasi infinite genomic space? To systematically examine this question in an up-to-date, multigenome comparison, we screened millions of primate transposed Alu SINE elements for incidences of homoplasious precise insertions and deletions. In genome-wide analyses, we identified and manually verified nine cases of precise parallel Alu insertions of apparently identical elements at orthologous positions in two ape lineages and twelve incidences of precise deletions of previously established SINEs. Correspondingly, eight precise parallel insertions and no exact deletions were detected in a comparison of lemuriform primate and human insertions spanning the range of primate diversity. With an overall frequency of homoplasious Alu insertions of only 0.01% (for human-chimpanzee-rhesus macaque) and 0.02-0.04% (for human-bushbaby-lemurs) and precise Alu deletions of 0.001-0.002% (for human-chimpanzee-rhesus macaque), real homoplasy is not considered to be a quantitatively relevant source of evolutionary noise. Thus, presence/absence patterns of Alu retrotransposons and, presumably, all LINE1-mobilized elements represent indeed the virtually homoplasy-free markers they are considered to be. Therefore, ancestral incomplete lineage sorting and hybridization remain the only serious sources of conflicting presence/absence patterns of retrotransposon insertions, and as such are detectable and quantifiable. [Homoplasy; precise deletions; precise parallel insertions; primates; retrotransposons.].

Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing

BackgroundMany applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ.ResultsIn the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions.ConclusionsNHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.