Cas9 Plasmid Research Articles

A Versatile Nonviral Delivery System for Multiplex Gene-Editing in the Liver.

Recent advances in CRISPR present attractive genome-editing toolsets for therapeutic strategies at the genetic level. Here, a liposome-coated mesoporous silica nanoparticle (lipoMSN) is reported as an effective CRISPR delivery system for multiplex gene-editing in the liver. The MSN provides efficient loading of Cas9 plasmid as well as Cas9 protein/guide RNA ribonucleoprotein complex (RNP), while liposome-coating offers improved serum stability and enhanced cell uptake. Hypothesizing that loss-of-function mutation in the lipid-metabolism-related genes pcsk9, apoc3, and angptl3 would improve cardiovascular health by lowering blood cholesterol and triglycerides, the lipoMSN is used to deliver a combination of RNPs targeting these genes. When targeting a single gene, the lipoMSN achieved a 54% gene-editing efficiency, besting the state-of-art Lipofectamine CRISPRMax. For multiplexing, lipoMSN maintained significant gene-editing at each gene target despite reduced dosage of target-specific RNP. By delivering combinations of targeting RNPs in the same nanoparticle, synergistic effects on lipid metabolism are observed in vitro and vivo. These effects, such as a 50% decrease in serum cholesterol after 4 weeks of post-treatment with lipoMSN carrying both pcsk9 and angptl3-targeted RNPs, could not be reached with a single gene-editing approach. Taken together, this lipoMSN represents a versatile platform for the development of efficient, combinatorial gene-editing therapeutics.

A CRISPR/Cas9-mediated gene knockout system in Aspergillus luchuensis mut. kawachii.

We developed an approach to genome editing of the white koji fungus, Aspergillus luchuensis mut. kawachii using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Co-transformation of AMA1-based Cas9 and gRNA expression plasmids achieved efficient gene knockout in A. kawachii. The plasmids were easily lost when selective pressure was removed, allowing for successive rounds of genome editing.

Efficient Genome Editing in Bacillus licheniformis Mediated by a Conditional CRISPR/Cas9 System.

Bacillus licheniformis is widely used to produce multiple enzymes and chemicals in industrial fermentation. It is also an organism that is hard to genetically manipulate, which is mainly attributed to its extremely low transformation efficiency. The lack of genetic modification technology severely limits its further application. In this study, an all-in-one conditional clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 plasmid was developed for B. licheniformis with the cas9 gene under the control of a xylose-inducible promoter. By means of this design, the expression of the cas9 gene could be repressed without xylose, which significantly improved the transformation ratio from less than 0.1 cfu/μg to 2.42 cfu/μg DNA. Compared with this conditional system, a constitutive overexpression system led to significant growth retardation in bacterial cells. Both the biomass and specific growth rate decreased greatly. After transformation, successful genome editing could be triggered by 0.5% xylose. When the α-amylase gene amyL was used as a genomic target, the efficiencies of its disruption using three different protospacer-adjacent motif (PAM) sequences were 64.3%, 70.9%, and 47.1%, respectively. Moreover, temperature plays a pivotal role in the function of the constructed CRISPR system. The maximum success rate reached 97% at 20 °C, while higher temperatures negatively impacted the function of the system. These results suggested that the design with a cas9 gene under the strict control of a xylose-inducible promoter significantly improved the success rate of genome editing in this host. This work contributes to the development of genetic manipulation and furthers the use of B. licheniformis as an efficient industrial workhorse.

High-copy genome integration of 2,3-butanediol biosynthesis pathway in Saccharomyces cerevisiae via in vivo DNA assembly and replicative CRISPR-Cas9 mediated delta integration

CRISPR Cas9 system is becoming an emerging genome-editing platform and has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. In this study, we developed a novel replicative and integrative CRISPR Cas9 genome-editing platform for large DNA construct in vivo assembly, replication, and high-copy genome integration in Saccharomyces cerevisiae. It harnessed advantages of autonomous replicative sequence in S. cerevisiae, in vivo DNA assembly, CRISPR Cas9, and delta integration. Enhanced green fluorescent protein was used as a marker to confirm large DNA construct in vivo assembly and genome integration. Based on this platform, an efficient 2,3- BDO producing yeast strain was rapidly constructed with up to 25-copy genome integration of 2,3-BDO biosynthesis pathway. Further strain engineering was conducted by multiplex disruption of ADH1, PDC1, PDC5 and MTH1 using a 2μ-based replicative CRISPR Cas9 plasmid containing donor DNAs. As a result, the 2,3-BDO titer was improved by 3.9 folds compared to that obtained by the initially engineered yeast and 50.5 g/L 2,3-BDO was produced by the final engineered yeast strain 36aS5-CFBDO in fed-batch fermentation without strain evolution and process optimization. This study demonstrated that the new replicative and integrative CRISPR Cas9 genome-editing platform was promising in generating an efficient 2,3-BDO-producing S. cerevisiae strain.

Dual sgRNA-directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system.

Ganoderma lucidum is an important medicinal mushroom in traditional Chinese medicine. However, the lack of adequate genetic tools has hindered molecular genetic research in and the genetic modification of this species. Here, we report that the presence of an intron is necessary for the efficient expression of the heterologous phosphinothricin‐resistance and green fluorescent protein genes in G. lucidum. Moreover, we improved the CRISPR/Cas9‐mediated gene disruption frequency in G. lucidum by adding an intron upstream of the Cas9 gene. Our results showed that the disruption frequency of the orotidine 5’‐monophosphate decarboxylase gene (ura3) in transformants containing the glyceraldehyde‐3‐phosphate dehydrogenase gene intron in the Cas9 plasmid is 14–18 in 107 protoplasts, which is 10.6 times higher than that in transformants without any intron sequence. Furthermore, genomic fragment deletions in the ura3 and GL17624 genes were achieved via a dual sgRNA‐directed CRISPR/Cas9 system in G. lucidum. We achieved a ura3 deletion frequency of 36.7% in G. lucidum. The developed method provides a powerful platform to generate gene deletion mutants and will facilitate functional genomic studies in G. lucidum.

Near-infrared optogenetic engineering of photothermal nanoCRISPR for programmable genome editing

We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.

Mutations in NlInR1 affect normal growth and lifespan in the brown planthopper Nilaparvata lugens

The brown planthopper (BPH) Nilaparvata lugens contains two insulin receptor homologues, designated NlInR1 and NlInR2. NlInR1 is strikingly homologous to the typical InR in insects and vertebrates, containing a ligand-activated intracellular tyrosine kinase catalytic domain. Herein, we report an optimized CRISPR/Cas9 system to induce mutations in the NlInR1 locus in BPH, consisting of a Cas9 plasmid that is specifically expressed in the germline via the Nlvasa promoter and versatile sgRNA expression plasmids under the control of the U6 promoter. We systematically evaluated the efficiency of injection mix compositions and demonstrated an appropriate combination of Cas9/sgRNA to target essential genes. Furthermore, we showed that homozygous mutants for the NlInR1 gene are early embryonic lethal, whereas heterozygous mutants grow more slowly, exhibit a severe reduction in body weight and wing size and live longer than the wild type. Interestingly, the severity of the mutant phenotype was different when targeting distinct important domains of the NlInR1 locus. The severity of the mutant phenotype is similar to that of insulin/insulin-like growth factor (IGF) signaling pathway deficiencies in vertebrates, suggesting a conserved function of NlInR1 in the regulation of development and longevity. Global expression profiling suggests that NlInR1 regulates many cellular processes in BPH, including insulin resistance, phototransduction, metabolism, endocytosis, longevity, biosynthesis and protein processing. Our results also pave the way for understanding the precise molecular mechanism of insulin signaling in wing polyphenism in insects.

643P - Impact of CCL4 knockout using CRISPR Cas-9 technology on colorectal tumour progression

BackgroundThe Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 system (CRISPR/Cas9 system), is the defense mechanism in Streptococcus pyogenes against viruses, now it is widely used by researchers as a precise gene editing tool that allows a targeted genomic editing with the least possible off targets. Colorectal cancer (CRC) has recently become a devastating disease with mortality rate exceeded 9% in 2018, where the survival rate worsens once it metastasizes to vital organs such as the liver or lungs. Chemokines are one of the key players that promote cancer cell metastasis in some types of cancers, including colorectal cancer, where increasing evidence shows that CRC accumulates invasive and metastatic capacities through tumor microenvironment. In the light of this, we aimed at understanding the tumor microenvironment to achieve a comprehensive therapy, aiming in preventing early metastasis. MethodsMicroarray was done by our research group on early metastasized CRC cells to liver in a group of rats with CRC. In silico sgRNA CRISPR designing tools were used to design a sgRNA targeting C-C motif chemokine ligand 4 (CCL4) gene. The insert was ligated into a Cas9 expressing plasmid and transformed into E.coli. The Plasmid was then extracted and sequenced. CRC cells were transfected with the cloned plasmid. Functional assays were performed. ResultsMicroarray results showed that CCL4 was highly expressed in early metastasized CRC cells to liver that gives a hypothesis that it has a role in cancer progression. Sequencing results confirmed a successful sgRNA insertion into the Cas9 plasmid. In addition, knocking out the expression of CCL4 in CRC cells significantly inhibited the cellular metabolic activity compared to cells transfected with empty plasmid. Furthermore, the ability of CRC cells to proliferate and to form colonies was dramatically decreased in response to CCL4 knockout in comparison to that transfected with empty plasmid. ConclusionsCCL4 has oncogenic effect on CRC by its positive effect on cell proliferation properties and metabolic activity. The results indicate that CCL4 may act as a promising target for further studies to reveal its downstream impact on CRC progression. Legal entity responsible for the studyHassan Adwan, German University in Cairo, Cairo, Egypt. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

ADRB2 Gene Knockout in Human Primary T Cells by Multiple sgRNAs Construced using CRISPR/Cas9 Technology

To knockout ADRB2 gene rapidly and efficiently in human primary T cells by using CRISPR/Cas9 technology and multiple sgRNAs strategy. Six paired-sgRNAs, which were designed to target the 5' constitutive coding exons of ADRB2 gene, were cloned into pGL3-U6-sgRNA-PGK-Puro vector separately. The expre-ssion vectors containing the single sgRNAs were constructed and transiently co-transfected into HEK-293T cell line with Cas9 expression vector. The sgRNA-mediated cleavage efficiency was tested by T7EN I digestion assay. Concatenating four highly efficient paired sgRNAs were cloned into pGL3-U6-sgRNA-ccdB-EF1α-Puro expression vector. The reco-mbinant plasmid allows the cells to express 4 sgRNAs, which target different sites on the ADRB2 genomic locus. The cleavage efficiency and mutation model were tested by T7EN I digest assay and T-A cloning technique. Multiple sgRNAs plasmid and Cas9 plasmid was transiently transferred into human primary T cells by electroporation. Flow cytometry (FCM) was used to detect the knockout efficiency of β2 adrenergic receptor (β2-AR). The results of T7EN I digestion and TA cloning sequencing showed that the multiple sgRNAs strategy could obtain more abundant mutation types and higher gene editing efficiency than single sgRNA. In addition to the deletion and insertion of bases, large fragment DNA deletions and inversions could be observed. All of the random 10 TA clones for detection were genetically modified, thus the mutation efficiency was as high as 100%. FCM assay showed that 43.09% of the cells in the control T cells were β2-AR positive, but the proportion of β2-AR positive cells in the multiple sgRNAs electrotransformed T cells decreased to 25.61%. A method, which is simple and operable, for knocking out β2-AR in human primary T cells has been established preliminarily. The results are helpful for the further study of the role of β2-AR in human T cells.

Efficient generation of GHR knockout Bama minipig fibroblast cells using CRISPR/Cas9-mediated gene editing.

Dwarfism, also known as growth hormone deficiency (GHD), is a disease caused by genetic mutations that result in either a lack of growth hormone or insufficient secretion of growth hormone, resulting in a person's inability to grow normally. In the past, many studies focusing on GHD have made use of models of other diseases such as metabolic or infectious diseases. A viable GHD specific model system has not been used previously, thus limiting the interpretation of GHD results. The Bama minipig is unique to Guangxi province and has strong adaptability and disease resistance, and an incredibly short stature, which is especially important for the study of GHD. In addition, studies of GHR knockout Bama minipigs and GHR knockout Bama minipig fibroblast cells generated using CRISPR/Cas9 have not been previously reported. Therefore, the Bama minipig was selected as an animal model and as a tool for the study of GHD in this work. In this study, a Cas9 plasmid with sgRNA targeting the first exon of the GHR gene was transfected into Bama minipig kidney fibroblast cells to generate 22 GHR knockout Bama minipig kidney fibroblast cell lines (12 male monoclonal cells and 10 female monoclonal cells). After culture and identification, 11 of the 12 male clone cell lines showed double allele mutations, and the rate of positive alteration of GHR was 91.67%. Diallelic mutation of the target sequence occurred in 10 female clonal cell lines, with an effective positive mutation rate of 100%. Our experimental results not only showed that CRISPR/Cas9 could efficiently be used for gene editing in Bama minipig cells but also identified a highly efficient target site for the generation of a GHR knockout in other porcine models. Thus, the generation of GHR knockout male and female Bama fibroblast cells could lay a foundation for the birth of a future dwarfism model pig. We anticipate that the "mini" Bama minipig will be of improved use for biomedical and agricultural scientific research and for furthering our understanding of the genetic underpinnings of GHD.

Abstract 2647: MUC13 interacts with SOX2 leading to cell stemness and Anoikis resistance in colorectal cancer

Abstract Colorectal Cancer(CRC) is one of the leading cause of cancer related death in the United States today in both men and women. With an expected 50,260 deaths in 2017 it is the second leading cause of cancer related deaths among men and the third among women in the United states. Often in colorectal cancer it is not the primary tumor that is the killer, but it is the metastatic sites that develop in the later stages of the disease that is the true killer. About 90% of all cancer related deaths are due to the development of metastatic sites in the body. What is metastasis? Metastasis is the transfer of disease from the primary tumor site to another unconnected site within the body through a process called the metastatic cascade. Metastasis develops slowly over time, due to it being an inefficient process having to overcome several natural defenses within the body. One of these defenses is Anoikis, cell death due to lack of attachment to the extracellular matrix. The development of Anoikis resistant is key in the process of metastasis, because of the need of the cells to detach and circulate the body before settling at a new site within the body. The inhibition of Anoikis resistance development in CRC could lead to improve patient outcome and the failure of metastasis to develop in the lifetime of the disease in the patient. Thus, characterizing and developing methods of inhibition of Anoikis resistance could lead to the decrease in cancer related deaths in the United States as well as finding effect therapeutic options. One of the key characteristics of Anoikis resistance in cells is the develop of stem cell like characteristics, and expression of key stemness factors of Sox2, Oct4, and NANOG. The expression of these factors allows for the cells to overcome their need to be attached to the ECM and travel through the blood stream. We have found through analysis that the membrane bound muicin MUC13 interacts with Sox2 increasing cell stemness leading to the development of anoikis resistance in Colorectal Cancer. Cell cycle analysis of Sw620 cells with high endogenous MUC13 expression showed an 80% less cell death when compare to isogenic Sw480 cells after 48 hours. Western blot anaylsis of the time points 0,24,36, and 48 hrs showed an increase in Sox2 expression and MUC13 expression in Sw620 cells during 24 and 36hrs. To further understand this relationship MUC13 knockdown and overexpressing cells lines will be analyzed to see if any there is a change in the Anoikis phenotype of Sw620 and Sw480 cells. This will also be done with Sox2 knockdown and overexpression. We will also test different therapeutic options, specifically a novel non-viral delivery of MUC13 Crispr Cas9 plasmid to see it effect on Anoikis resistances. Citation Format: Kyle Doxtater, Chidi Zacheaus, Fatemeh Keramatnia, Mehdi Chaib, Manish Triparthi, Subhash Chauhan. MUC13 interacts with SOX2 leading to cell stemness and Anoikis resistance in colorectal cancer [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 2647.

Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9.

Purpose: Gene therapy has become a promising remedy to treat disease by modifying the person’s genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability.Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency. Results: Among the designed CLs, the four-component formula [DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum.Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.

Escherichia coli vectors having stringently repressible replication origins allow a streamlining of Crispr/Cas9 gene editing

Readily curable plasmids facilitate the construction of plasmid-free bacterial strains after the plasmid encoded genes are no longer needed. The most popular of these plasmids features a temperature-sensitive (Ts) pSC101 origin of replication which can readily revert during usage and cannot be used to construct Ts mutations in essential genes. Plasmid pAM34 which contains an IPTG-dependent origin of replication largely overcomes this issue but is limited by carrying the most commonly utilized antibiotic selection and replication origin. This study describes the construction of an expanded series of plasmid vectors having replication origins of p15a, RSF1030 or RSF1031 that like pAM34 have IPTG-dependent replication. Surprisingly, these plasmids can be cured in fewer generations than pAM34. Derivatives of pAM34 with alternative antibiotic selection markers were also constructed. The utility of these vectors is demonstrated in the construction of a CRISPR-Cas9 system consisting of an IPTG-dependent Cas9 plasmid and a curable guide RNA plasmid having a streptomycin counterselection marker. This system was successfully demonstrated by construction of point mutations, deletions and insertions in the E. coli genome with a very high efficiency and in a shorter timescale than extant methods. The plasmids themselves were readily cured either together or singly from the resultant strains with minimal effort.

Edition of TFAM gene by CRISPR/Cas9 technology in bovine model.

The mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA) binding protein essential for the initiation of transcription and genome maintenance. Recently it was demonstrated that the primary role of TFAM is to maintain the integrity of mtDNA and that it is a key regulator of mtDNA copy number. It was also shown that TFAM plays a central role in the mtDNA stress-mediated inflammatory response. In our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology in bovine fibroblasts, as TFAM regulates the replication specificity of mtDNA. We further attempted to maintain these cells in culture post edition in a medium supplemented with uridine and pyruvate to mimic Rho zero cells that are capable of surviving without mtDNA, because it is known that the TFAM gene is lethal in knockout mice and chicken. Moreover, we evaluated the effects of TFAM modification on mtDNA copy number. The CRISPR gRNA was designed to target exon 1 of the bovine TFAM gene and subsequently cloned. Fibroblasts were transfected with Cas9 and control plasmids. After 24 h of transfection, cells were analyzed by flow cytometry to evaluate the efficiency of transfection. The site directed-mutation frequency was assessed by T7 endonuclease assay, and cell clones were analyzed for mtDNA copy number by Sanger DNA sequencing. We achieved transfection efficiency of 51.3%. We selected 23 successfully transformed clones for further analysis, and seven of these exhibited directed mutations at the CRISPR/Cas9 targeted site. Moreover, we also found a decrease in mtDNA copy number in the gene edited clones compared to that in the controls. These TFAM gene mutant cells were viable in culture when supplemented with uridine and pyruvate. We conclude that this CRISPR/Cas9 design was efficient, resulting in seven heterozygous mutant clones and opening up the possibility to use these mutant cell lines as a model system to elucidate the role of TFAM in the maintenance of mtDNA integrity.

CRISPR/Cas9 mutagenesis in Volvox carteri

Volvox carteri and other volvocine green algae comprise an excellent model for investigating developmental complexity and its origins. Here we describe a method for targeted mutagenesis in V.carteri using CRISPR/Cas9 components expressed from transgenes. We used V.carteri nitrate reductase gene (nitA) regulatory sequences to conditionally express Streptococcus pyogenes Cas9, and V.carteri U6 RNA gene regulatory sequences to constitutively express single-guide RNA (sgRNA) transcripts. Volvox carteri was bombarded with both Cas9 vector and one of several sgRNA vectors programmed to target different test genes (glsA, regA and invA), and transformants were selected for expression of a hygromycin-resistance marker present on the sgRNA vector. Hygromycin-resistant transformants grown with nitrate as sole nitrogen source (inducing for nitA) were tested for Cas9 and sgRNA expression, and for the ability to generate progeny with expected mutant phenotypes. Some transformants of a somatic regenerator (Reg) mutant strain receiving sgRNA plasmid with glsA protospacer sequence yielded progeny (at a rate of ~0.01%) with a gonidialess (Gls) phenotype similar to that observed for previously described glsA mutants, and sequencing of the glsA gene in independent mutants revealed short deletions within the targeted region of glsA, indicative of Cas9-directed non-homologous end joining. Similarly, bombardment of a morphologically wild-type strain with the Cas9 plasmid and sgRNA plasmids targeting regA or invA yielded regA and invA mutant transformants/progeny, respectively (at rates of 0.1-100%). The capacity to make precisely directed frameshift mutations should greatly accelerate the molecular genetic analysis of development in V.carteri, and of developmental novelty in the volvocine algae.

Fluorinated Acid‐Labile Branched Hydroxyl‐Rich Nanosystems for Flexible and Robust Delivery of Plasmids

Nucleic acid-based therapy specially needs a safe and robust delivery vector. Herein, a novel fluorinated acid-labile branched hydroxyl-rich polycation (ARP-F) is proposed for the flexible and effective delivery nanovector of different plasmids including reporter genes and the Cas9 plasmid. Acid-responsive polycation (ARP) with plentiful ortho ester linkages and hydroxyl groups is first synthesized via a facile one-pot ring-opening polymerization, followed by decoration of fluorinated alkyl chains onto ARP to achieve ARP-F. ARP-F possesses good pH-responsive degradability, biocompatibility, and its preliminary transfection ability evaluated by reporter plasmids pRL-CMV (encoding Renilla luciferase) and pEGFP-N1 (encoding enhanced green fluorescent protein) is also excellent. CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9) technology is a potent genome-editing tool. The subsequent delivery of pCas9-surv (one typical all-in-one Cas9 plasmid) mediated by ARP-F exhibits impressive in vitro and in vivo tumor inhibition performances. In addition, the combination of ARP-F/pCas9-surv with temozolomide could further enhance tumor inhibition activities by increasing the sensitivity of cancer cells to anticancer drugs. Such high-performance polycation would provide a very promising means to produce efficient delivery nanovectors of versatile plasmids.

Abstract 2153: Development of a novel MEK mutation driven drug resistant melanoma model by CRISPR/Cas9 technology

Abstract Melanoma remains the most lethal form of skin cancer exhibiting high mortality rates due to a high likelihood of developing metastases and acquiring drug resistance. Approximately 40-50% of melanomas contain oncogenic BRAF mutations of which 75-90% harbor the BRAFV600E mutation. This mutation constitutively activates the mitogen-activating protein kinase (MAPK) signaling pathway leading to uncontrolled cell growth and oncogenesis. Recent combination therapies of BRAF and MEK specific inhibitors have shown improved progression-free patient response in phase II clinical trials. However, in the majority of patients, acquired resistance to MAPK pathway inhibitor therapies develops after approximately 12 months of treatment. Preclinical studies have suggested that MEK1 mutations confer resistance to BRAF and MEK inhibitors. In this study, we used the CRISPR genome editing technology to generate a drug resistant MEK1Q56P knock-in mutation within the A375 melanoma cell line which naturally harbors the BRAFV600E mutation. We validated this new isogenic cell model using both molecular and biofunctional approaches. Method and results: Single guide RNAs (sgRNAs) were designed and built into Cas9 plasmids to bind and cut desired regions in the MEK1 target region. The melanoma cell line A375 was co-transfected with single guide-containing Cas9 plasmids and donor plasmids. Single cells were cloned and expanded for subsequent screening of the desired gene mutation events. The introduction of the MEK1Q56P mutation in the cells was then confirmed via Sanger sequencing and NGS at the genetic level. Validation of the mutation in mRNA transcripts was carried out by RT-PCR followed by sequencing. The cell line maintains permanent and genetically stable resistance characteristics during cell culture expansion without the use of selective pressure. Drug responses to BRAF and MEK1 specific inhibitors and non-specific chemotherapy drugs were compared between the A375 MEK1Q56P isogenic cell line and the parental cell line in 2D and 3D culture environments. Results demonstrated that the isogenic MEK1Q56P cell line showed significant and specific resistance to BRAF inhibitors in comparison to the parental A375 line. Using gene editing we have targeted an endogenous loci within a melanoma cell line creating a novel in-vitro model to aid in basic and translational melanoma research as well as drug screening efforts. This new approach to cell line development provides direct in vitro bio-functional evidence of a drug resistant gene driving tumor cells survival under targeted anti-cancer treatments. Furthermore, this A375 MEK1Q56P isogenic cell line represents a new type of drug resistance model that contains a defined genetic resistance mechanism. This model provides an invaluable tool for developing next generation therapeutics that can overcome drug resistance in melanoma. Citation Format: Lysa-Anne Volpe, Metewo Selase Enuameh, Luping Chen, Elizabeth Turner, John Foulke, Fang Tian. Development of a novel MEK mutation driven drug resistant melanoma model by CRISPR/Cas9 technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2153.

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute.

CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid‐based CRISPR/Cas9 system. To tackle this, a self‐assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid‐guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9‐mediated E7 knockout leads to significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine‐based therapeutics.

Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing

The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii.