Preclinical toxicity analyses of lentiviral vectors expressing the HIV-1 LTR-specific designer-recombinase Brec1.

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.

Nucleolar protein TAAP1/C22orf46 confers pro-survival signaling in non-small cell lung cancer.

Tumor cells subvert immune surveillance or lytic stress by harnessing inhibitory signals. Hence, bispecific antibodies have been developed to direct CTLs to the tumor site and foster immune-dependent cytotoxicity. Although applied with success, T cell-based immunotherapies are not universally effective partially because of the expression of pro-survival factors by tumor cells protecting them from apoptosis. Here, we report a CRISPR/Cas9 screen in human non-small cell lung cancer cells designed to identify genes that confer tumors with the ability to evade the cytotoxic effects of CD8+ T lymphocytes engaged by bispecific antibodies. We show that the gene C22orf46 facilitates pro-survival signals and that tumor cells devoid of C22orf46 expression exhibit increased susceptibility to T cell-induced apoptosis and stress by genotoxic agents. Although annotated as a non-coding gene, we demonstrate that C22orf46 encodes a nucleolar protein, hereafter referred to as "Tumor Apoptosis Associated Protein 1," up-regulated in lung cancer, which displays remote homologies to the BH domain containing Bcl-2 family of apoptosis regulators. Collectively, the findings establish TAAP1/C22orf46 as a pro-survival oncogene with implications to therapy.

Activation of recombinases at specific DNA loci by zinc-finger domain insertions.

Recombinases have several potential advantages as genome editing tools compared to nucleases and other editing enzymes, but the process of engineering them to efficiently recombine predetermined DNA targets demands considerable investment of time and labor. Here we sought to harness zinc-finger DNA-binding domains (ZFDs) to program recombinase binding by developing fusions, in which ZFDs are inserted into recombinase coding sequences. By screening libraries of hybrid proteins, we optimized the insertion site, linker length, spacing and ZFD orientation and generated Cre-type recombinases that remain dormant unless the insertionally fused ZFD binds its target site placed in the vicinity of the recombinase binding site. The developed fusion improved targeted editing efficiencies of recombinases by four-fold and abolished measurable off-target activity in mammalian cells. The ZFD-dependent activity is transferable to a recombinase with relaxed specificity, providing the means for developing fully programmable recombinases. Our engineered recombinases provide improved genome editing tools with increased precision and efficiency.

Discovery and characterization of novel Cre-type tyrosine site-specific recombinases for advanced genome engineering.

Tyrosine-type site-specific recombinases (Y-SSRs) are versatile tools for genome engineering due to their ability to mediate excision, integration, inversion and exchange of genomic DNA with single nucleotide precision. The ever-increasing need for sophisticated genome engineering is driving efforts to identify novel SSR systems with intrinsic properties more suitable for particular applications. In this work, we develop a systematic computational workflow for annotation of putative Y-SSR systems and apply this pipeline to identify and characterize eight new naturally occurring Cre-type SSR systems. We test their activity in bacterial and mammalian cells and establish selectivity profiles for the new and already established Cre-type SSRs with regard to their ability to mutually recombine their target sites. These data form the basis for sophisticated genome engineering experiments using combinations of Y-SSRs in research fields including advanced genomics and synthetic biology. Finally, we identify putative pseudo-sites and potential off-targets for Y-SSRs in the human and mouse genome. Together with established methods for altering the DNA-binding specificity of this class of enzymes, this work should facilitate the use of Y-SSRs for future genome surgery applications.

CRISPR/Cas9 Screen in Gastric Cancer Patient-Derived Organoids Reveals KDM1A-NDRG1 Axis as a Targetable Vulnerability.

Viability CRISPR screens have proven indispensable in parsing genome function. However, their application in new, more physiologically relevant culturing systems like patient-derived organoids (PDOs) has been much slower. To probe epigenetic contribution to gastric cancer (GC), the third leading cause of cancer-related deaths worldwide, the first negative selection CRISPR screen in GC PDOs that faithfully preserve primary tumor characteristics is performed. Extensive quality control measurements showing feasibility of CRISPR screens in primary organoid culture are provided. The screen reveals the histone lysine demethylase-1A (KDM1A) to constitute a GC vulnerability. Both genetic and pharmacological inhibition of KDM1A cause organoid growth retardation. Further, it is shown that most of KDM1A cancer-supporting functions center on repression of N-myc downstream regulates gene-1 (NDRG1). De-repression of NDRG1 by KDM1A inhibitors (KDM1Ai) causes inhibition of Wnt signaling and a strong G1 cell cycle arrest. Finally, by profiling 20 GC PDOs, it is shown that NDRG1 upregulation predicts KDM1Ai response with 100% sensitivity and 82% specificity in the tested cohort. Thus, this work pioneers the use of negative selection CRISPR screens in patient-derived organoids, identifies a marker of KDM1Ai response, and accordingly a cohort of patients who may benefit from such therapy.

Precise excision of HTLV-1 provirus with a designer-recombinase.

The human T cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus that persists as a provirus in the genome of infected cells and can lead to adult T cell leukemia (ATL). Worldwide, more than 10 million people are infected and approximately 5% of these individuals will develop ATL, a highly aggressive cancer that is currently incurable. In the last years, genome editing tools have emerged as promising antiviral agents. In this proof-of-concept study, we use substrate-linked directed evolution (SLiDE) to engineer Cre-derived site-specific recombinases to excise the HTLV-1 proviral genome from infected cells. We identified a conserved loxP-like sequence (loxHTLV) present in the long terminal repeats of the majority of virus isolates. After 181 cycles of SLiDE, we isolated a designer-recombinase (designated RecHTLV), which efficiently recombines the loxHTLV sequence in bacteria and human cells with high specificity. Expression of RecHTLV in human Jurkat T cells resulted in antiviral activity when challenged with an HTLV-1 infection. Moreover, expression of RecHTLV in chronically infected SP cells led to the excision of HTLV-1 proviral DNA. Our data suggest that recombinase-mediated excision of the HTLV-1 provirus represents a promising approach to reduce proviral load in HTLV-1-infected individuals, potentially preventing the development of HTLV-1-associated diseases.

Prediction of designer-recombinases for DNA editing with generative deep learning.

Site-specific tyrosine-type recombinases are effective tools for genome engineering, with the first engineered variants having demonstrated therapeutic potential. So far, adaptation to new DNA target site selectivity of designer-recombinases has been achieved mostly through iterative cycles of directed molecular evolution. While effective, directed molecular evolution methods are laborious and time consuming. Here we present RecGen (Recombinase Generator), an algorithm for the intelligent generation of designer-recombinases. We gather the sequence information of over one million Cre-like recombinase sequences evolved for 89 different target sites with which we train Conditional Variational Autoencoders for recombinase generation. Experimental validation demonstrates that the algorithm can predict recombinase sequences with activity on novel target-sites, indicating that RecGen is useful to accelerate the development of future designer-recombinases.

RNAi-Mediated Screen of Primary AML Cells Nominates MDM4 as a Therapeutic Target in NK-AML with DNMT3A Mutations.

DNA-methyltransferase 3A (DNMT3A) mutations belong to the most frequent genetic aberrations found in adult acute myeloid leukemia (AML). Recent evidence suggests that these mutations arise early in leukemogenesis, marking leukemic progenitors and stem cells, and persist through consolidation chemotherapy, providing a pool for AML relapse. Currently, there are no therapeutic approaches directed specifically against this cell population. To unravel therapeutically actionable targets in mutant DNMT3A-driven AML cells, we have performed a focused RNAi screen in a panel of 30 primary AML samples, all carrying a DNMT3A R882 mutation. As one of the strongest hits, we identified MDM4 as a gene essential for proliferation of primary DNMT3AWT/R882X AML cells. We analyzed a publicly available RNA-Seq dataset of primary normal karyotype (NK) AML samples and found a trend towards MDM4 transcript overexpression particularly in DNMT3A-mutant samples. Moreover, we found that the MDM2/4 inhibitor ALRN-6924 impairs growth of DNMT3AWT/R882X primary cells in vitro by inducing cell cycle arrest through upregulation of p53 target genes. Our results suggest that MDM4 inhibition is a potential target in NK-AML patients bearing DNMT3A R882X mutations.

Correction of a Factor VIII genomic inversion with designer-recombinases.

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.

The Paf1 complex positively regulates enhancer activity in mouse embryonic stem cells.

The RNA polymerase II (RNAPII) associated factor 1 complex (Paf1C) plays critical roles in modulating the release of paused RNAPII into productive elongation. However, regulation of Paf1C-mediated promoter-proximal pausing is complex and context dependent. In fact, in cancer cell lines, opposing models of Paf1Cs' role in RNAPII pause-release control have been proposed. Here, we show that the Paf1C positively regulates enhancer activity in mouse embryonic stem cells. In particular, our analyses reveal extensive Paf1C occupancy and function at super enhancers. Importantly, Paf1C occupancy correlates with the strength of enhancer activity, improving the predictive power to classify enhancers in genomic sequences. Depletion of Paf1C attenuates the expression of genes regulated by targeted enhancers and affects RNAPII Ser2 phosphorylation at the binding sites, suggesting that Paf1C-mediated positive regulation of pluripotency enhancers is crucial to maintain mouse embryonic stem cell self-renewal.

MLLT6 maintains PD-L1 expression and mediates tumor immune resistance.

Tumor cells subvert immune surveillance by harnessing signals from immune checkpoints to acquire immune resistance. The protein PD-L1 is an important component in this process, and inhibition of PD-L1 elicits durable anti-tumor responses in a broad spectrum of cancers. However, immune checkpoint inhibition that target known pathways is not universally effective. A better understanding of the genetic repertoire underlying these processes is necessary to expand our knowledge in tumor immunity and to facilitate identification of alternative targets. Here, we present a CRISPR/Cas9 screen in human cancer cells to identify genes that confer tumors with the ability to evade the cytotoxic effects of the immune system. We show that the transcriptional regulator MLLT6 (AF17) is required for efficient PD-L1 protein expression and cell surface presentation in cancer cells. MLLT6 depletion alleviates suppression of CD8+ cytotoxic T cell-mediated cytolysis. Furthermore, cancer cells lacking MLLT6 exhibit impaired STAT1 signaling and are insensitive to interferon-γ-induced stimulation of IDO1, GBP5, CD74, and MHC class II genes. Collectively, our findings establish MLLT6 as a regulator of oncogenic and interferon-γ-associated immune resistance.

Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors.

In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient editing of human induced pluripotent stem cells (iPSC). Whereas we were unable to correct a disease-causing mutation in patient derived iPSCs using a CRISPR/Cas9 nuclease approach, we corrected the mutation back to wild type with high efficiency utilizing an adenine BE. We also used adenine and cytosine BEs to introduce nine different cancer associated TP53 mutations into human iPSCs with up to 90% efficiency, generating a panel of cell lines to investigate the biology of these mutations in an isogenic background. Finally, we pioneered the use of prime editing in human iPSCs, opening this important cell type for the precise modification of nucleotides not addressable by BEs and to multiple nucleotide exchanges. These approaches eliminate the necessity of deriving disease specific iPSCs from human donors and allows the comparison of different disease-causing mutations in isogenic genetic backgrounds.

A heterodimer of evolved designer-recombinases precisely excises a human genomic DNA locus.

Site-specific recombinases (SSRs) such as the Cre/loxP system are useful genome engineering tools that can be repurposed by altering their DNA-binding specificity. However, SSRs that delete a natural sequence from the human genome have not been reported thus far. Here, we describe the generation of an SSR system that precisely excises a 1.4 kb fragment from the human genome. Through a streamlined process of substrate-linked directed evolution we generated two separate recombinases that, when expressed together, act as a heterodimer to delete a human genomic sequence from chromosome 7. Our data indicates that designer-recombinases can be generated in a manageable timeframe for precision genome editing. A large-scale bioinformatics analysis suggests that around 13% of all human protein-coding genes could be targetable by dual designer-recombinase induced genomic deletion (dDRiGD). We propose that heterospecific designer-recombinases, which work independently of the host DNA repair machinery, represent an efficient and safe alternative to nuclease-based genome editing technologies.

CRISPR/Cas9 as a tool to dissect cancer mutations.

The CRISPR/Cas9 system is transforming many biomedical disciplines, including cancer research. Through its flexible programmability and efficiency to induce DNA double strand breaks it has become straightforward to introduce cancer mutations into cells in vitro and/or in vivo. However, not all mutations contribute equally to tumorigenesis and distinguishing essential mutations for tumor growth and survival from biologically inert mutations is cumbersome. Here we present a method to screen for the functional relevance of mutations in high throughput in established cancer cell lines. We employ the CRISPR/Cas9 system to probe cancer vulnerabilities in a colorectal carcinoma cell line in an attempt to identify novel cancer driver mutations. We designed 100 high quality sgRNAs that are able to specifically cleave mutations present in the colorectal carcinoma cell line RKO. An all-in-one lentiviral library harboring these sgRNAs was then generated and used in a pooled screen to probe possible growth dependencies on these mutations. Genomic DNA at different time points were collected, the sgRNA cassettes were PCR amplified, purified and sgRNA counts were quantified by means of deep sequencing. The analysis revealed two sgRNAs targeting the same mutation (UTP14A: S99delS) to be depleted over time in RKO cells. Validation and characterization confirmed that the inactivation of this mutation impairs cell growth, nominating UTP14A: S99delS as a putative driver mutation in RKO cells. Overall, our approach demonstrates that the CRISPR/Cas9 system is a powerful tool to functionally dissect cancer mutations at large-scale.

Comparative RNAi Screens in Isogenic Human Stem Cells Reveal SMARCA4 as a Differential Regulator.

Large-scale RNAi screens are a powerful approach to identify functions of genes in a cell-type-specific manner. For model organisms, genetically identical (isogenic) cells from different cell types are readily available, making comparative studies meaningful. However, large-scale screens in isogenic human primary cells remain challenging. Here, we show that RNAi screens are possible in genetically identical human stem cells, using induced pluripotent stem cells as intermediates. The screens revealed SMARCA4 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 4) as a stemness regulator, while balancing differentiation distinctively for each cell type. SMARCA4 knockdown in hematopoietic stem and progenitor cells caused impaired self-renewal in vitro and in vivo with skewed myeloid differentiation; whereas, in neural stem cells, it impaired self-renewal while biasing differentiation toward neural lineage, through combinatorial SWI/SNF subunit assembly. Our findings pose a powerful approach for deciphering human stem cell biology and attribute distinct roles to SMARCA4 in stem cell maintenance.

STK3 is a therapeutic target for a subset of acute myeloid leukemias.

Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation and accumulation of immature myeloblasts, which impair normal hematopoiesis. While this definition categorizes the disease into a distinctive group, the large number of different genetic and epigenetic alterations actually suggests that AML is not a single disease, but a plethora of malignancies. Still, most AML patients are not treated with targeted medication but rather by uniform approaches such as chemotherapy. The identification of novel treatment options likely requires the identification of cancer cell vulnerabilities that take into account the different genetic and epigenetic make-up of the individual tumors. Here we show that STK3 depletion by knock-down, knock-out or chemical inhibition results in apoptotic cells death in some but not all AML cell lines and primary cells tested. This effect is mediated by a premature activation of cyclin dependent kinase 1 (CDK1) in presence of elevated cyclin B1 levels. The anti-leukemic effects seen in both bulk and progenitor AML cells suggests that STK3 might be a promising target in a subset of AML patients.

The long noncoding RNA lncR492 inhibits neural differentiation of murine embryonic stem cells.

RNA interference (RNAi) screens have been shown to be valuable to study embryonic stem cell (ESC) self-renewal and they have been successfully applied to identify coding as well as noncoding genes required for maintaining pluripotency. Here, we used an RNAi library targeting >640 long noncoding RNAs (lncRNA) to probe for their role in early cell differentiation. Utilizing a Sox1-GFP ESC reporter cell line, we identified the lncRNA lncR492 as lineage-specific inhibitor of neuroectodermal differentiation. Molecular characterization showed that lncR492 interacts with the mRNA binding protein HuR and facilitates its inhibitory function by activation of Wnt signaling. Thus, lncRNAs modulate the fate decision of pluripotent stem cells.

lncRNA Panct1 Maintains Mouse Embryonic Stem Cell Identity by Regulating TOBF1 Recruitment to Oct-Sox Sequences in Early G1.

Long noncoding RNAs (lncRNAs) have been implicated in diverse biological processes, including embryonic stem cell (ESC) maintenance. However, their functional mechanisms remain largely undefined. Here, we show that the lncRNA Panct1 regulates the transient recruitment of a putative X-chromosome-encoded protein A830080D01Rik, hereafter referred to as transient octamer binding factor 1 (TOBF1), to genomic sites resembling the canonical Oct-Sox motif. TOBF1 physically interacts with Panct1 and exhibits a cell-cycle-specific punctate localization in ESCs. At the chromatin level, this correlates with its recruitment to promoters of pluripotency genes. Strikingly, mutating an octamer-like motif in Panct1 RNA abrogates the strength of TOBF1 localization and recruitment to its targets. Taken together, our data reveal a tightly controlled spatial and temporal pattern of lncRNA-mediated gene regulation in a cell-cycle-dependent manner and suggest that lncRNAs might function as barcodes for identifying genomic addresses for maintaining cellular states.

Development of a genetic sensor that eliminates p53 deficient cells.

The TP53 gene fulfills a central role in protecting cells from genetic insult. Given this crucial role it might be surprising that p53 itself is not essential for cell survival. Indeed, TP53 is the single most mutated gene across different cancer types. Thus, both a theoretical and a question of significant practical applicability arise: can cells be programmed to make TP53 an essential gene? Here we present a genetic p53 sensor, in which the loss of p53 is coupled to the rise of HSV-TK expression. We show that the sensor can distinguish both p53 knockout and cells expressing a common TP53 cancer mutation from otherwise isogenic TP53 wild-type cells. Importantly, the system is sensitive enough to specifically target TP53 loss-of-function cells with the HSV-TK pro-drug Ganciclovir both in vitro and in vivo. Our work opens new ways to programming cell intrinsic transformation protection systems that rely on endogenous components.

ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator.

Telomeres constitute the ends of linear chromosomes and together with the shelterin complex form a structure essential for genome maintenance and stability. In addition to the constitutive binding of the shelterin complex, other direct, yet more transient interactions are mediated by the CST complex and HOT1/HMBOX1, while subtelomeric variant repeats are recognized by NR2C/F transcription factors. Recently, the Kruppel-like zinc finger protein ZBTB48/HKR3/TZAP has been described as a novel telomere-associated factor in the vertebrate lineage. Here, we show that ZBTB48 binds directly both to telomeric and to subtelomeric variant repeat sequences. ZBTB48 is found at telomeres of human cancer cells regardless of the mode of telomere maintenance and it acts as a negative regulator of telomere length. In addition to its telomeric function, we demonstrate through a combination of RNAseq, ChIPseq and expression proteomics experiments that ZBTB48 acts as a transcriptional activator on a small set of target genes, including mitochondrial fission process 1 (MTFP1). This discovery places ZBTB48 at the interface of telomere length regulation, transcriptional control and mitochondrial metabolism.