Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR.

Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10-5 bp-1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity - an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation.

During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1-binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins.

The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells.

CRISPR-Cas12a nucleases have expanded the toolbox for targeted genome engineering in a broad range of organisms. Here, using a high-throughput engineering approach, we explored the potential of a novel CRISPR-MAD7 system for genome editing in human cells. We evaluated several thousand optimization conditions and demonstrated accurate genome reprogramming with modified MAD7. We identified crRNAs that allow for ≤95% non-homologous end joining (NHEJ) and 66% frameshift mutations in various genes and observed the high-cleavage fidelity of MAD7 resulting in undetectable off-target activity. We explored the dsDNA delivery efficiency of CRISPR-MAD7, and by using our optimized transfection protocol, we obtained ≤85% chimeric antigen receptor (CAR) insertions in primary T cells, thus exceeding the baseline integration efficiencies of therapeutically relevant transgenes using currently available virus-free technologies. Finally, we evaluated multiplex editing efficiency with CRISPR-MAD7 and demonstrated simultaneous ≤35% CAR transgene insertions and ≤80% gene disruption efficiencies. Both the platform and our transfection procedure are easily adaptable for further preclinical studies and could potentially be used for clinical manufacturing of CAR T cells.

CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells.

Discovery of genomic safe harbor sites (SHSs) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CARs), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in induced pluripotent stem cells (iPSCs), primary T and natural killer (NK) cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC toward CD34+ hematopoietic stem and progenitor cells (HSPCs), lymphoid progenitor cells (LPCs), and NK cells and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHSs can be used to engineer cells for adoptive immunotherapies.

The general control nonderepressible-2 kinase mediates stress response and longevity induced by target of rapamycin inactivation in Caenorhabditis elegans.

The general control nonderepressible 2 (GCN2) kinase is a nutrient-sensing pathway that responds to amino acids deficiency and induces a genetic program to effectively maintain cellular homeostasis. Here we established the conserved role of Caenorhabditis elegans GCN-2 under amino acid limitation as a translation initiation factor 2 (eIF2) kinase. Using a combination of genetic and molecular approaches, we showed that GCN-2 kinase activity plays a central role in survival under nutrient stress and mediates lifespan extension conferred by dietary restriction (DR) or inhibition of the major nutrient-sensing pathway, the target of rapamycin (TOR). We also demonstrated that the GCN-2 and TOR signaling pathways converge on the PHA-4/FoxA transcription factor and its downstream target genes to ensure survival of the whole organism under a multitude of stress conditions, such as nutrient scarcity or environmental stresses. This is one step forward in the understanding of evolutionary conserved mechanisms that confer longevity and healthspan.

Domains involved in TAF15 subcellular localisation: dependence on cell type and ongoing transcription.

TAF15 (TBP associated factor 15) is a member of the highly conserved TET (also known as FET) protein family of RNA binding proteins (RBP), which comprises in addition FUS (fused in sarcoma, also known as TLS, translocated in liposarcoma) and EWS (Ewing sarcoma protein). The TET proteins are implied to play important roles in the onset of specific tumours, certain forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In this study we identified the domains of TAF15 responsible for its subcellular localisation in human (HeLa) cells and experimentally confirmed the presence of a transportin-dependent nuclear localisation signal (NLS) at its carboxy-terminus. We demonstrated that additional domains of TAF15 contributed, albeit to a less prominent extent, to its subcellular localisation. In the carboxy-terminus we identified an arginine and glycine rich (RGG) domain, capable of being targeted to stress granules. We, moreover, showed that TAF15 cellular localisation depended on ongoing transcription and that independent domains of TAF15 engaged in nucleolar capping upon transcription inhibition. Finally, we demonstrated that TAF15 localisation was differentially regulated in the HeLa and the neuronal HT22 cell lines and that TAF15 co-localised with a minor subset of RNA granules in the cytoplasm of HT22 cells, supporting a model whereupon TAF15 plays a role in RNA transport and/or local RNA translation.