CRISPR-Cas9 Mediated Genome Editing Research Articles

Abstract A084: CDK12 inhibition drives mitotic catastrophe, triggering genome instability and innate immune response in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited therapies. In our screening using patient-derived organoids, we identified cyclin-dependent kinase 12 (CDK12) as a promising target in a subset of PDAC. Both pharmacological and genetic inhibition of CDK12 exerted potent antitumor activity in vitro and in vivo, inducing double-strand DNA breaks and apoptosis. CDK12 is a member of cyclin-dependent kinases that is involved in gene expression regulations at multiple levels. Recently, CDK12 has gained attention as a therapeutic target in several types of cancers including triple-negative breast cancer and Ewing sarcoma. However, little is known about the impact and possibility of CDK12 inhibition in pancreatic cancer treatment. As the lack of specific inhibitors that selectively target CDK12, studying CDK12 has long been challenging. To overcome this, we used CRISPR-Cas9 mediated genome editing to generate FKBP12F36V (dTAG) knock-in PDAC cell lines, enabling rapid elimination of endogenous CDK12 using a degrader against dTAG (dTAG-13). Quantitative-image-based cytometry and time lapse imaging showed that CDK12 ablation induces mitotic arrest, DNA damage and apoptosis (mitotic catastrophe). Specifically, loss of CDK12 kinase activity in the S phase was sufficient to slow DNA replication speed, leading to spindle assembly checkpoint activation and mitotic catastrophe. Importantly, cells that escaped mitotic death accumulate micronuclei, triggering immune response signaling that is mediated by the cGAS-STING-type I interferon pathway. Our results suggest that CDK12 is a promising target in a subset of PDAC patients, where CDK12 inhibition exerts antitumor activity both in cell autonomous and non-cell autonomous ways through inducing mitotic catastrophe as well as triggering immune response. Citation Format: Dosuke Iwadate, Keisuke Yamamoto, Hiroyuki Kato, Keisuke Tateishi, Mitsuhiro Fujishiro. CDK12 inhibition drives mitotic catastrophe, triggering genome instability and innate immune response in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A084.

CRISPR-Cas9 mediated genome editing of Kluyveromyces marxianus for iterative, multiplexed gene disruption and pathway integration.

Kluyveromyces marxianus, a thermotolerant, fast-growing, Crabtree-negative yeast, is a promising chassis for the manufacture of various bioproducts. Although several genome editing tools are available for this yeast, these tools still require refinement to enable more convenient and efficient genetic modification. In this study, we engineered the K. marxianus NBRC 104275 strain by impairing the nonhomologous end joining and enhancing the homologous recombination machinery, which resulted in improved homology-directed repair effective on homology arms of up to 40 bp in length. Additionally, we simplified the CRISPR-Cas9 editing system by constructing a strain for integrative expression of Cas9 nuclease and plasmids bearing different selection markers for gRNA expression, thereby facilitating iterative genome editing without the need for plasmid curing. We demonstrated that tRNA was more effective than the hammerhead ribozyme for processing gRNA primary transcripts, and readily assembled tRNA-gRNA arrays were used for multiplexed editing of at least four targets. This editing tool was further employed for simultaneous scarless in vivo assembly of a 12-kb cassette from three fragments and marker-free integration for expressing a fusion variant of fatty acid synthase, as well as the integration of genes for starch hydrolysis. Together, the genome editing tool developed in this study makes K. marxianus more amenable to genetic modification and will facilitate more extensive engineering of this nonconventional yeast for chemical production.

Lipid droplet accumulation in Wdr45-deficient cells caused by impairment of chaperone-mediated autophagic degradation of Fasn

Backgroundβ-Propeller protein-associated neurodegeneration (BPAN) is a genetic neurodegenerative disease caused by mutations in WDR45. The impairment of autophagy caused by WDR45 deficiency contributes to the pathogenesis of BPAN; however, the pathomechanism of this disease is largely unknown. Lipid dyshomeostasis is involved in neurogenerative diseases, but whether lipid metabolism is affected by Wdr45 deficiency and whether lipid dyshomeostasis contributes to the progression of BPAN are unclear.MethodsWe generated Wdr45 knockout SN4741 cell lines using CRISPR‒Cas9-mediated genome editing, then lipid droplets (LDs) were stained using BODIPY 493/503. Chaperone-mediated autophagy was determined by RT-qPCR and western blotting. The expression of fatty acid synthase (Fasn) was detected by western blot in the presence or absence of the lysosomal inhibitor NH4Cl and the CMA activator AR7. The interaction between Fasn and HSC70 was analyzed using coimmunoprecipitation (Co-IP) assay. Cell viability was measured by a CCK-8 kit after treatment with the Fasn inhibitor C75 or the CMA activator AR7.ResultsDeletion of Wdr45 impaired chaperone-mediated autophagy (CMA), thus leading to lipid droplet (LD) accumulation. Moreover, Fasn can be degraded via CMA, and that defective CMA leads to elevated Fasn, which promotes LD formation. LD accumulation is toxic to cells; however, cell viability was not rescued by Fasn inhibition or CMA activation. Inhibition of Fasn with a low concentration of C75 did not affect cell viability but decreases LD density.ConclusionsThese results suggested that Fasn is essential for cell survival but that excessive Fasn leads to LD accumulation in Wdr45 knockout cells.

CRISPR-Cas9 mediated genome editing in soybean for improving quality traits

CRISPR-Cas9 mediated genome editing in soybean for improving quality traits

Disruption of a licorice cellulose synthase-derived glycosyltransferase gene demonstrates its in planta role in soyasaponin biosynthesis

Key messageCRISPR–Cas9-mediated disruption of a licorice cellulose synthase-derived glycosyltransferase gene, GuCSyGT, demonstrated the in planta role of GuCSyGT as the enzyme catalyzing 3-O-glucuronosylation of triterpenoid aglycones in soyasaponin biosynthesis.Triterpenoid glycosides (saponins) are a large, structurally diverse group of specialized metabolites in plants, including the sweet saponin glycyrrhizin produced by licorice (Glycyrrhiza uralensis) and soyasaponins that occur widely in legumes, with various bioactivities. The triterpenoid saponin biosynthetic pathway involves the glycosylation of triterpenoid sapogenins (the non-sugar part of triterpenoid saponins) by glycosyltransferases (GTs), leading to diverse saponin structures. Previously, we identified a cellulose synthase-derived GT (CSyGT), as a newly discovered class of triterpenoid GT from G. uralensis. GuCSyGT expressed in yeast, which could transfer the sugar glucuronic acid to the C3 position of glycyrrhetinic acid and soyasapogenol B, which are the sapogenins of glycyrrhizin and soyasaponin I, respectively. This suggested that GuCSyGT is involved in the biosynthesis of glycyrrhizin and soyasaponin I. However, the in planta role of GuCSyGT in saponin biosynthesis remains unclear. In this study, we generated GuCSyGT-disrupted licorice hairy roots using CRISPR–Cas9-mediated genome editing and analyzed the saponin content. This revealed that soyasaponin I was completely absent in GuCSyGT-disrupted lines, demonstrating the in planta role of GuCSyGT in saponin biosynthesis.

The Influence of TET2 and/or DNMT3A Knockout on the 5hmC Levels and Growth Characteristics of Modified Human Primary CD4 T Cells after Activation

Angioimmunoblastic T-cell lymphoma (AITL) has a unique immunohistological profile featuring malignant CD4+ T follicular helper cells (Tfh) inter-mixed with an extensive infiltrate of multiple lineages of immune cells. The AITL tumor microenvironment (TME) classically features small or large B cells (variably Epstein-Barr Virus (EBV) positive), reactive CD8+ T-cells, plasma cells and proliferative high endothelial venules (HEVs). Common genetic mutations have been identified within the malignant Tfh clone in AITL including epigenetic modifiers TET2, DNMT3A, and IDH2. DNMT3A mutations in AITL occur at a frequency of 20-38.5%and most of the DNMT3A mutations occur together with TET2 mutations. Tet2/ Dnmt3a double-knockout mice show synergistic effects on the expression of lineage-specifying transcription factor genes via epigenetic mechanisms. However, it is still not clear how DNMT3A mutations enhance the effect of TET2 mutations on promoting AITL development. To investigate how TET2 and DNMT3A mutations interact in AITL development, we first generated TET2 and/or DNMT3A knockout human primary CD4-T cells with CRISPR-Cas9 mediated genome editing. TET proteins could oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmc) in replication- independent DNA demethylation. Unmethylated cytosine base can be methylated by the DNA methylation machinery (DNMT3A/B) to form 5mC, which in turn provides the substrate for TET enzymes to produce 5hmC. TET and DNMT family members are thus needed to be dynamically modulate the epigenome to maintain cellular homeostasis or alter cellular function or development. We found that in response to CD3/CD28 stimulation, WT CD4-T cells exhibited division mediated dilutional reduction in 5 hmC which was later reconstitution. However, in TET2 or DKO cells, this process was compromised, showing delayed global 5hmc levels recovery (Fig. 1A). We then investigated whether the changes in methylation dynamics would alter cellular function. When using CD3/CD28 beads stimulation (10 days/cycle), we found that after two cycles of stimulation, these CD4-T cells with different gene editing started showing distinct behaviors with proliferation and apoptosis when exposed to CD3/CD28 beads again. The TET2/ DNMT3A double knockout (DKO) CD4-T cells showed more rapid response in cell division and higher proliferation rate than the other three groups (Fig. 1B). Compared with WT cells, consistently higher apoptotic cells were found in DNMT3A-KO group, but not with either TET2-KO or DKO cells (Fig. 1C). Interestingly, withdraw of beads at the end of the 10 days cycle, DKO cells kept higher baseline proliferation level than WT cells (Fig. 1D). We then used autologous or allogeneic EBV transformed B-cell for stimulation of the CD4 T-cells after one or two cycles of CD3/28 beads stimulation, and different response kinetics were observed among these prior activated CD4-T cells with TET2 and/or DNMT3A mutations. DKO cells showed more rapid response in cell division and elevated percentages of replicated cells to either autologous or allogeneic B-cells compared with WT cells (Fig. 1E). An interesting aspect of this B/T interaction is that, even the autologous B-cells could activate a large percentage of the T-cell population, particularly in the DKO cells, so it is not likely due to specific antigen recognition but rather a non-specific stimulation by the B-cells, probably via one or more co-stimulatory molecules (ICOSL, CD86 or 4-1BBL). This may be relevant to in vivo evolution of the mutated T-cells with B-cells/activated B-cells providing support.

Deficiency of ligase IV leads to reduced NHEJ, accumulation of DNA damage, and can sensitize cells to cancer therapeutics

Ligase IV is a key enzyme involved during DNA double-strand breaks (DSBs) repair through nonhomologous end joining (NHEJ). However, in contrast to Ligase IV deficient mouse cells, which are embryonic lethal, Ligase IV deficient human cells, including pre-B cells, are viable. Using CRISPR-Cas9 mediated genome editing, we have generated six different LIG4 mutants in cervical cancer and normal kidney epithelial cell lines. While the LIG4 mutant cells showed a significant reduction in NHEJ, joining mediated through microhomology-mediated end joining (MMEJ) and homologous recombination (HR) were significantly high. The reduced NHEJ joining activity was restored by adding purified Ligase IV/XRCC4. Accumulation of DSBs and reduced cell viability were observed in LIG4 mutant cells. LIG4 mutant cells exhibited enhanced sensitivity towards DSB-inducing agents such as ionizing radiation (IR) and etoposide. More importantly, the LIG4 mutant of cervical cancer cells showed increased sensitivity towards FDA approved drugs such as Carboplatin, Cisplatin, Paclitaxel, Doxorubicin, and Bleomycin used for cervical cancer treatment. These drugs, in combination with IR showed enhanced cancer cell death in the background of LIG4 gene mutation. Thus, our study reveals that mutation in LIG4 results in compromised NHEJ, leading to sensitization of cervical cancer cells towards currently used cancer therapeutics.

CRISPR‐Cas9 Genome Editing in the Moss Physcomitrium (Formerly Physcomitrella) patens

Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR-Cas9 mediated genome editing, P. patens was unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR-Cas9, molecular manipulations in P. patens have greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vector Alternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectors Basic Protocol 2: Designing the oligonucleotide-based homology-directed repair (HDR) template Alternate Protocol 2: Designing the plasmid-based HDR template Basic Protocol 3: Inducing genome editing by transforming CRISPR vector into P. patens protoplasts Basic Protocol 4: Identifying edited plants.

CRISPR CAS-9 Mediated Genome Editing and One Health Related Diseases

CRISPR CAS-9 Mediated Genome Editing and One Health Related Diseases

CRISPR-Cas9 mediated genome editing of Huntington's disease neurospheres.

Huntington's disease (HD) is a fatal genetic disease caused by polyglutamine aggregation encoded by an expanded CAG repeat in the huntingtin gene (HTT). In this study, we cultured neurospheres derived from R6/2 mice, a representative animal model of HD, as an in vitro model. GuideRNAs were designed to induce large deletion or frameshift indel mutation of CAG expansion. These gRNAs and Cas9 were delivered to the R6/2 neurospheres and disease-related phenotypes were observed. Deletion or indel mutation of the CAG repeat was confirmed by PCR, T7E1 assay and sequencing of the edited neurospheres. Edited neurospheres showed decreased polyglutamine aggregation compared with control HD neurospheres. In the edited neurosphere, we confirmed the upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and brain-derived neurotrophic factor (BDNF), whose reduced expressions are closely involved in the disease progression. In addition, flow cytometry result showed an increase in cell viability with an overall decrease in necrotic and apoptotic populations among edited R6/2 neurospheres. Additional siRNA experiments confirmed that the increased viability was decreased through inhibition of PGC-1α or BDNF. Our study confirmed that CAG repeat of R6/2 mouse-derived neurospheres can be edited through CRISPR-Cas9. Editing of CAG repeat sequence decreases polyglutamine aggregation and cellular apoptosis of HD neurospheres, which may be related to the increased expressions of PGC-1α and BDNF.Our data provide the evidence that CRISPR-Cas9 mediated genome editing has therapeutic potential on HD neuronal cells.

A Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants

Plant expression vectors are essential tools for gene functional analysis and molecular plant breeding. The gene of interest is transferred to the vector by molecular cloning technology. Nimble Cloning is a newly developed molecular cloning method with the advantages of simplicity, efficiency, and standardization. In this study, we developed a “pNC” vector system that contains 55 Nimble Cloning-compatible vectors for functional analysis of genes in plants. These vectors contain the NC frame flanked by unique adapters for one-step and standardized Nimble Cloning. We demonstrate that the pNC vectors are convenient and effective for the functional analysis of plant genes, including the study of gene ectopic expression, protein subcellular localization, protein–protein interaction, gene silencing (RNAi), virus-induced gene silencing, promoter activity, and CRISPR–Cas9-mediated genome editing. The “pNC” vector system represents a high-throughput toolkit that can facilitate the large-scale analysis of plant functional genomics.

Construction of tomato plants with suppressed endo-β-N-acetylglucosaminidase activity using CRISPR-Cas9 mediated genome editing

High mannose-type free N-glycans with a single N-acetyl-D-glucosamine (GlcNAc) residue at the reducing end (GN1-HMT-FNGs) are produced by cytosolic endo-β-N-acetylglucosaminidase (EC:3.2.1.96) (ENGase) and are ubiquitous in differentiating and growing plant cells. To elucidate the physiological functions of HMT-FNGs in plants, we identified the ENGase gene in tomato (Solyc06g050930) and detected ENGase activity and increased production of GN1-HMT-FNGs during tomato fruit maturation. However, the precise role of GN1-HMT-FNGs in fruit maturation remains unclear. In this study, we established tomato ENGase mutants with suppressed ENGase activity via CRISPR/Cas9 genome editing technology. DNA sequencing of the Δeng mutants (T0 and T1 generations) revealed that they had the same mutations in the genomic DNA around the target sequences. Three null CRISPR/Cas9 segregant plants of the T1 generation (Δeng1-2, −22, and −26) were used to measure ENGase activity and analyze the structural features of HMT-FNGs in the leaves. The Δeng mutants did not exhibit ENGase activity and produced GN2-HMT-FNGs bearing tow GlcNAc residues at the reducing end side instead of GN1-HMT-FNGs. The Δeng mutants lack the N-terminal region of ENGase, indicating that the N-terminal region is important for full ENGase activity. The fruits of Δeng mutants (T2 generation) also showed loss of ENGase activity and similar structural features of HMT-FNGs of the T1 generation. However, there was no significant difference in fruit maturation between the T2 generation of the Δeng mutants and the wild type. The Δeng mutants rich in GN2-HMT-FNGs could be offered as a new tomato that is different from wild type containing GN1-HMT-FNGs.

A straightforward plant prime editing system enabled highly efficient precise editing of rice Waxy gene

CRISPR Cas9-mediated genome editing is highly efficient at targeted site-specific gene knock-out through NHEJ (Non-Homology End Joining), but ineffective for specific DNA integration through HDR (Homology Directed Repair) for precise gene editing. Base editors can make limited base substitutions but only within restricted small windows of the protospacer. Prime editing has been applied in plants with various degrees of success. However, several questions such as low and inconsistent editing efficiencies across different target sites need to be addressed. We compared two prime editing approaches PE3 and PE2 at two neighboring target sites within rice Waxy gene to partially address those questions. A straightforward PE2 plant prime editing system retrofitted from a regular CRISPR-Cas9 editing system can deliver highly efficient up to 66.7% precise gene editing. Various forms of precise editing including base substitutions, small deletions and insertions can be accurately achieved. The secondary structure variations of different pegRNAs may be the primary reason for inconsistent editing across different target sites and should be the optimization focus to further improve plant prime editing.

Shear stress switches the association of endothelial enhancers from ETV/ETS to KLF transcription factor binding sites

Endothelial cells (ECs) lining blood vessels are exposed to mechanical forces, such as shear stress. These forces control many aspects of EC biology, including vascular tone, cell migration and proliferation. Despite a good understanding of the genes responding to shear stress, our insight into the transcriptional regulation of these genes is much more limited. Here, we set out to study alterations in the chromatin landscape of human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress. To do so, we performed ChIP-Seq for H3K27 acetylation, indicative of active enhancer elements and ATAC-Seq to mark regions of open chromatin in addition to RNA-Seq on HUVEC exposed to 6 h of laminar shear stress. Our results show a correlation of gained and lost enhancers with up and downregulated genes, respectively. DNA motif analysis revealed an over-representation of KLF transcription factor (TF) binding sites in gained enhancers, while lost enhancers contained more ETV/ETS motifs. We validated a subset of flow responsive enhancers using luciferase-based reporter constructs and CRISPR-Cas9 mediated genome editing. Lastly, we characterized the shear stress response in ECs of zebrafish embryos using RNA-Seq. Our results lay the groundwork for the exploration of shear stress responsive elements in controlling EC biology.

Discovery of potent and versatile CRISPR-Cas9 inhibitors engineered for chemically controllable genome editing.

Anti-CRISPR (Acr) proteins are encoded by many mobile genetic elements (MGEs) such as phages and plasmids to combat CRISPR–Cas adaptive immune systems employed by prokaryotes, which provide powerful tools for CRISPR–Cas-based applications. Here, we discovered nine distinct type II-A anti-CRISPR (AcrIIA24–32) families from Streptococcus MGEs and found that most Acrs can potently inhibit type II-A Cas9 orthologs from Streptococcus (SpyCas9, St1Cas9 or St3Cas9) in bacterial and human cells. Among these Acrs, AcrIIA26, AcrIIA27, AcrIIA30 and AcrIIA31 are able to block Cas9 binding to DNA, while AcrIIA24 abrogates DNA cleavage by Cas9. Notably, AcrIIA25.1 and AcrIIA32.1 can inhibit both DNA binding and DNA cleavage activities of SpyCas9, exhibiting unique anti-CRISPR characteristics. Importantly, we developed several chemically inducible anti-CRISPR variants based on AcrIIA25.1 and AcrIIA32.1 by comprising hybrids of Acr protein and the 4-hydroxytamoxifen-responsive intein, which enabled post-translational control of CRISPR–Cas9-mediated genome editing in human cells. Taken together, our work expands the diversity of type II-A anti-CRISPR families and the toolbox of Acr proteins for the chemically inducible control of Cas9-based applications.

Elucidating the Importance and Regulation of Key Enhancers for Human MEIS1 Expression

Myeloid ecotropic virus insertion site 1 (MEIS1) is essential for normal hematopoiesis and is deregulated in a large subset of acute myeloid leukemia (AML) by yet unknown mechanisms. We previously identified 3 candidate enhancer regions: enhancer region 1 (E1) at -2 kb upstream; enhancer region 2 (E2) at +10.6 kb downstream inside intron 6; and enhancer region 3 (E3) +140 kb downstream of the translation start site. In the current study, we utilized CRISPR-Cas9 genome editing to further characterize these enhancers in a human AML cell line and identify the key transcription factors (TFs) associated with their function.To efficiently track MEIS1 expression levels, a GFP reporter, a P2A self-cleaving peptide tag and a hemagglutinin tag at its translation start site was introduced in a MEIS1 high expressing human AML cell line, U937. Then we introduced random mutations (Indels) along the MEIS1 locus utilizing a CRISPR-Cas9 mediated genome editing vector system in mono-allelic MEIS1-GFP-tagged U937 cells with special focus on the previously identified enhancer regions to find the key sequences important to the function of the MEIS1 enhancer regions. Two targeted regions yielding the highest proportion of GFP - cells corresponded to the E2 enhancer region within intron 6 and were referred to as E2.1 and E2.2. Using chromosome conformation capture (3C) assay, we detected a significantly decreased interaction (p=0.0022) between the promoter and the intron 6 region surrounding the E2 region in E2.2 targeted cells compared to the parental cells. Moreover, our data indicated that the DNA sequence within E2.2 is highly critical to this region's enhancer function which is further influenced by the larger genomic region surrounding the E2.1 gRNA targeted site.To identify TFs binding to the E2 region, we further scrutinized the E2.2 indel region for loss of TF binding sites. We performed TF prediction analysis and performed a protein pull down-mass spectrometry experiment to identify TF candidates. The overlap yielded a list of 7 TFs, each of which we targeted via CRISPR/Cas9. Reduction in GFP levels was only observed for FLI1 locus targeting but not for the other 6 TFs. Concordant reduction in MEIS1 and FLI1 levels were confirmed by immunoblotting. Additionally, chromatin immunoprecipitation (ChIP) followed by quantitative PCR revealed significant FLI1 enrichment at the promoter and at 3 sites surrounding the E2.2 region (p=0.0004) compared to 4 control regions scattered along the MEIS1 locus. Given a previous study indicating MEIS1 upregulation of FLI1 in normal hematopoiesis, we hypothesised that a positive feedback loop may exist between FLI1 and MEIS1 in AML. Since MEIS1 levels are frequently elevated in normal karyotype AML (CN-AML), we used the murine Hoxa9/Meis1 AML model as a surrogate for CN-AML and performed Meis1 ChIP-seq analysis. We detected direct Meis1 binding to the intronic region of the mouse Fli1 gene as well as other ETS factor loci, in Hoxa9/Meis1 cells.To better understand the clinical relevance of FLI1 in AML, we analyzed the Beat AML dataset. High FLI1 transcript levels correlated with adverse overall survival in CN-AML (p=0.044). Additionally, we observed a trend towards worse outcome with high FLI1 in the NPM1-mutated CN-AML subtype (p=0.069). We also observed a similar correlation in CN-AML for another ETS factor, ELF1, which we had previously shown to bind and upregulate MEIS1 expression in AML, suggesting a broader unrecognized role for ETS factors in AML.In summary, we have developed a rapid flow cytometry-based readout for the fine dissection and characterization of the cis-regulatory elements and associated TFs critical for MEIS1 transcription via CRISPR-Cas9 genetic manipulation. Our study revealed FLI1 as the candidate key regulator of MEIS1 expression and a positive correlation between FLI1 mRNA levels and worse overall survival in MEIS1-high AML subgroups. DisclosuresNo relevant conflicts of interest to declare.

Modeling High-Grade Serous Ovarian Carcinoma Using a Combination of In Vivo Fallopian Tube Electroporation and CRISPR-Cas9-Mediated Genome Editing.

Ovarian cancer is the most lethal gynecologic cancer to date. High-grade serous ovarian carcinoma (HGSOC) accounts for most ovarian cancer cases, and it is most frequently diagnosed at advanced stages. Here, we developed a novel strategy to generate somatic ovarian cancer mouse models using a combination of in vivo electroporation and CRISPR-Cas9-mediated genome editing. Mutation of tumor suppressor genes associated with HGSOC in two different combinations (Brca1, Tp53, Pten with and without Lkb1) resulted in successfully generation of HGSOC, albeit with different latencies and pathophysiology. Implementing Cre lineage tracing in this system enabled visualization of peritoneal micrometastases in an immune-competent environment. In addition, these models displayed copy number alterations and phenotypes similar to human HGSOC. Because this strategy is flexible in selecting mutation combinations and targeting areas, it could prove highly useful for generating mouse models to advance the understanding and treatment of ovarian cancer. SIGNIFICANCE: This study unveils a new strategy to generate genetic mouse models of ovarian cancer with high flexibility in selecting mutation combinations and targeting areas.

Generating Nonmosaic Mutants in Xenopus Using CRISPR-Cas in Oocytes.

In CRISPR-Cas9 genome editing, double-strand DNA breaks (DSBs) primarily undergo repair through nonhomologous end joining (NHEJ), which produces insertion or deletion of random nucleotides within the targeted region (indels). As a result, frameshift mutation-mediated loss-of-function mutants are frequently produced. An alternative repair mechanism, homology-directed repair (HDR), can be used to fix DSBs at relatively low frequency. By injecting a DNA-homology repair construct with the CRISPR-Cas components, specific nucleotide sequences can be introduced within the target region by HDR. We have taken advantage of the fact that Xenopus oocytes have much higher levels of HDR than eggs to increase the effectiveness of creating precise mutations. We introduced the oocyte host transfer technique, well established for knockdown of maternal mRNA for loss-of-function experiments, to CRISPR-Cas9-mediated genome editing. The host-transfer technique is based on the ability of Xenopus oocytes to be isolated, injected with CRISPR-Cas components, and cultured in vitro for up to 5 d before fertilization. During these 5 d, CRISPR-Cas components degrade, preventing further alterations to the paternal or maternal genomes after fertilization and resulting in heterozygous, nonmosaic embryos. Treatment of oocytes with a DNA ligase IV inhibitor, which blocks the NHEJ repair pathway, before fertilization further improves the efficiency of HDR. This method allows straightforward generation of either nonmosaic F0 heterozygous indel mutant Xenopus or Xenopus with efficient, targeted insertion of small DNA fragments (73-104 nt). The germline transmission of mutations in these animals allows homozygous mutants to be obtained one generation (F1) sooner than previously reported.

Disruption of CTCF Boundary Remodels Chromatin Domain and Reprograms HOX Gene Transcription in Acute Myeloid Leukemia

The organization of the mammalian genome into separate domains either facilitates or restricts interactions between regulatory elements, e.g. enhancers and promoters, for normal gene regulation. CTCF insulator constrains the temporal HOX gene expression patterns within the confined chromatin domain for normal development. However, dysregulation of HOX genes is also a common feature of acute myeloid leukemia (AML). The molecular mechanisms of aberrant HOX expression and associated AML pathogenesis remain unclear. Here, we found that CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) acts as a chromatin boundary to establish and maintain AML specific aberrant HOX gene expression patterns and is essential for pathogenesis of myeloid malignancies.To unbiasedly and systematically investigate the mechanism underlying HOX gene regulation in AML, we employed a pooled CRISPR-Cas9 genetic knockout library screening to interrogate intergenic regulatory and chromatin boundary elements in all 4 HOX gene loci. Through this screening, we identified the CBS7/9 chromatin boundary located between HOXA7 and HOXA9 genes that is critical for maintaining aberrant expression of posterior HOXA genes including oncogene HOXA9 and HOXA10 in primary AML patients and cells. To further confirm the role of CBS7/9 in HOX gene regulation, two single-guide RNAs (sgRNAs) flanking 47 base pairs (bps) of the core CTCF motif located between HOXA7 and HOXA9 geneswere employed to guild Cas9 for deletion of the CBS7/9 boundary in 3 different subtypes of AML cells containing MLL rearrangements or NPM1C+ mutation. The defected CBS7/9 resulted in a decrease in CTCF binding to the caudal HOXA domain and an inhibition of posterior HOXA gene expression.To further delineate the role of CBS7/9 chromatin boundary in HOX gene organization and regulation, we then carried out genome wide ChIP-seq, ATAC-seq, 4C, and RNA-seq analysis to examine the changes in chromatin domain organization and corresponding gene expression patterns compared wild-type control and the CBS7/9 KO AML cells. Disruption of CBS7/9 boundary resulted in a spreading of repressive H3K27me3 domain into the caudal HOXA domain that subsequently impaired AML associated active TAD domain by altering enhancer/promoter chromatin accessibility and reducing ectopic interactions among the posterior HOXA genes. Consistent with the role of CBS7/9 boundary in HOX locus chromatin structure and organization, impairment of CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid specific transcriptome profiles important for pathogenesis of myeloid malignancies suggesting that the CBS7/9 boundary mediated ectopic HOX expression patterns might be essential for pathogenesis of myeloid malignancies. To further test this possibility, the CBS 7/9 boundary was deleted in both MLL rearranged AML cells and primary patient samples by the CRISPR-Cas9 mediated genome editing and the edited cells were transplanted into NSG mice for the development of human AML in mice. Disruption of CBS chromatin boundary in the HOXA locus reduced human leukemic blast development and invasion as well as enhanced survival of both transplanted AML cell xenograft and patient derived xenograft (PDX) mouse models. Thus, our finding demonstrated that CTCF boundary not only constrains normal gene expression program for memory of cell identity, but also plays a role in maintaining oncogenic transcription program for leukemic transformation. Alteration of CTCF mediated topological boundaries in the oncogene loci provides therapeutic approaches for targeting oncogenic expression program and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Selection-Free Non-Viral Method Revealed Highly Efficient Crispr-Cas9 Genome Editing of Human Pluripotent Stem Cells Guided by Cellular Autophagy

CRISPR-Cas9 mediated genome editing of human pluripotent stem cells (hPSCs) provides strong avenues for human disease modeling, drug discovery and cell replacement therapy. Genome editing of hPSCs is an extremely inefficient process and requires complex gene delivery and selection methods to improve edit efficiency which are not ideal for clinical applications. Here, we have shown a selection free simple lipofectamine based transfection method where a single plasmid encoding guide RNA (gRNA) and Cas9 selectively transfected hPSCs at the colony edges. Upon dissection and sequencing, the edge cells showed more than 30% edit frequency compared to the reported 3% rate under no selections. Increased cellular health of the edge cells as revealed by reduced autophagy gene-expressions is critical for such transfection pattern. Edge specific transfection was inhibited by blocking lysosomal activity which is essential for autophagy. Hence, our method provides robust scarless genome-editing of hPSCs which is ideal for translational research.