Surveyor Assay Research Articles

Identify Myeloid Differentiation-Related MiRNAs Response to ATRA Induction by RNA Sequencing and CRISPR/Cas9 Gene Editing

To identify differentiation related miRNA and evaluate roles of miRNA during ATRA induced myeloid differentiation. The small RNA sequencing was used to analyze differential expressed miRNAs in ATRA induced NB4 cells. Then the several up or down-regulated miRNA were selected as the research candidates. SgRNAs targeting the genome of each miRNA were designed and NB4 cells with inducible expression of Cas9 protein were generated. After transduced sgRNA into NB4/Cas9 cells, the mutation level by PCR and surveyor assay were evaluated. The cell differentiation level was investigated by surface CD11b expression via flow cytometry. A total of 410 mature miRNAs which expressed in NB4 cells were detected out after treated by ATRA, 74 miRNAs were up-regulated and 55 were down-regulated miRNAs with DNA cleavage generated by CRISPR/Cas9 was assayed directly by PCR or surveyor assay, quantitative PCR showed that the expression of miRNA was downregulated, which evaluated that gene edition successfully inhibitied the expression of mature miRNA. MiR-223 knockout showed the myeloid differentation of NB4 significantly inhibitied, while miRNA-155 knockout showed the myeloid differentation of NB4 cells significantly increased. CRISPR/Cas9 is a powerful tool for gene editing and can lead to miRNA knockout. Knockouts of miR-223 and miR-155 have shown a differentiation-related phenotype, and the potential mechanism is the integrative regulation of target genes.

Erratum: CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy.

An erratum was issued for: Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells. The phrases"surveyor assay"and"SurveyorNuclease" havebeen updated to "T7E1 assay" to " T7endonuclease I" respectively. Step 1.2 inthe Protocol has been updated from: Surveyor nucleaseassay of sgRNA NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by surveyor nuclease assay (also known as T7 endonuclease I (T7EI) assay)17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon. to: T7 endonuclease assay of sgRNA NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by T7 endonuclease (T7EI)assay17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon. Figure 1 in the Representative Results has been updatedfrom: Figure 1: HMEJ-mediated targeted integration in vitro. (A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2acells. GFP+ cells were sorted at day 3 for surveyor assay. (C) Surveyor assay for Cdx2 targeting: 6 different sgRNAs were designed for surveyor assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.Please click here to view a larger version of this figure. to: Figure 1: HMEJ-mediated targeted integration in vitro. (A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2acells. GFP+ cells were sorted at day 3 for T7EIassay. (C) T7EI assay for Cdx2 targeting: 6 different sgRNAs were designed for T7EI assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.Please click here to view a larger version of this figure.

Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and Emulsion Coupling assay as a low-cellularity workflow for single-cell cloning.

The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (Surveyor assay), mRNA RT-PCR assessing altered gene expression, and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process.

Increased Efficiency for Biallelic Mutations of the CCR5 Gene by CRISPR-Cas9 Using Multiple Guide RNAs As a Novel Therapeutic Option for Human Immunodeficiency Virus.

CCR5 is a coreceptor of human immunodeficiency virus type 1 (HIV-1). Transplantation of hematopoietic stem cells homozygous for a 32-bp deletion in CCR5 resulted in a loss of detectable HIV-1 in two patients, suggesting that genetic strategies to knockout CCR5 expression would be a promising gene therapy approach for HIV-1-infected patients. In this study, we targeted CCR5 by CRISPR-Cas9 with a single-guide (sgRNA) and observed 35% indel frequency. When we expressed hCas9 and two gRNAs, the Surveyor assay showed that Cas9-mediated cleavage was increased by 10% with two sgRNAs. Genotype analysis on individual clones showed 11 of 13 carried biallelic mutations, where 4 clones had frameshift (FS) mutations. Taken together, these results indicate that the efficiency of biallelic FS mutations and the knockout of the CCR5 necessary to prevent viral replication were significantly increased with two sgRNAs. These studies demonstrate the knockout of CCR5 and the potential for translational development.

Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system

Primary familial brain calcification (PFBC) is a chronic progressive neurogenetic disorder. Its clinical symptoms mainly include dyskinesia, cognitive disorder and mental impairment; and the pathogenesis remains unclear. Studies have shown that SLC20A2 is the most common pathogenic gene of the disease. Since the Slc20a2 gene knockout mouse model could result in fetal growth restriction, in order to better understand the pathogenesis of PFBC, the present study used the CRISPR/Cas9 technology to construct a conditional knockout model of Slc20a2 gene in the striatum of mice. First, three sgRNAs (single guide RNAs) were designed to target the exon3 of Slc20a2 gene. The activity of the respective sgRNA was verified by constructing expression plasmids, transfecting cells and Surveyor assay. Second, the SgRNA with the highest activity was selected to generate the recombinant AAV-Cre virus, which was injected into the striatum of mice by stereotactic method. In vitro experiments showed that the three sgRNAs could effectively mediate Cas9 cleavage of the respective target DNA. The activity of Cre recombinase of the AAV-Cre was confirmed by immunofluorescence assay. Immunohistochemistry, TA clone, high-throughput sequencing and Western blot were used to detect and evaluate the efficiency of Slc20a2 gene knockout. The results showed that the Slc20a2 expression in the striatum of mice in the experimental group decreased significantly. In this study, three sgRNAs capable of knockout of Slc20a2 were successfully designed, and the conditional knockout of the Slc20a2 gene in the striatum of mouse was successfully established by the CRISPR/Cas9 technology, thereby providing an effective animal model for studying the pathogenesis of PFBC.

Generation of novel Il2rg-knockout mice with clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9.

X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.

Abnormal expression of Wnt3a and inhibiting role of its molecular-targeted intervening in hepatocellular carcinoma

Objective: To investigate the Wnt3a expression in tissues of HCC and its gene knockout on effects of HepG2 cell proliferation or xenograft tumor growth. Methods: Hepatic Wnt3a expressions in 87 HCC and their matched surrounding tissues were observed by tissue microarray and immunohistochemistry for analyzing its clinicopathological characteristics; Wnt3a-knockout HepG2 cell lines were established by Crispr/cas9-sgRNA system and genomic cleavage efficiency was verified at gene level by surveyor assay. The relative proteins were confirmed by Western blotting; Cell Counting Kit-8 assay was used to examine cell proliferation after knocking-out Wnt3a successfully, and the nude mice HepG2 cell xenograft tumors delete that the relationship between Wnt3a and HCC growth. Results: The positive Wnt3a with brown staining particles was mainly distributed in cytosol and membrane of hepatocytes. The incidence of hepatic Wnt3a expression in cancerous tissues (95.4%) was significantly higher (χ (2) = 47.754, P < 0.001) than that in their surrounding tissues (49.4%). The high Wnt3a expression was 70.1% in the HCC and only 14.9% in the surrounding tissues. High Wnt3a expression was associated with poorly-differentiated grade, liver cirrhosis, HBV infection, portal vein invasion, TNM stage and 5-year survival rate. After knocked-out by Crispr/cas9-sgRNA system successfully, Wnt3a expression was down-regulated significantly at gene or protein level. Key molecule β-catenin in cytoplasma was obviously inhibited. HepG2 cell lines proliferation was suppressed in time-dependent manner. The nude mice HepG2 cell xenograft tumors confirmed that the knock-out of Wnt3a could significantly supressed HCC growth with slower speed (t = 6.418, P < 0.001), smaller volume(869.4 ± 222.5 mm(3) vs 355.0 ± 99.9 mm(3), t = 5.168, P < 0.001), and lighter weight (0.88 ± 0.20 g vs 0.35 ± 0.11 g, t = 5.628, P < 0.001)compared with the control group. Conclusion: Abnormal expression of Wnt3a could be expected as a promising target for HCC gene therapy.

Genotyping Genetically Modified (GM) Mice.

Prior to generating a new mouse model, it is important to plan the method that will be used to detect which of the mice generated have the mutation(s) desired. Nearly, all types of mutations may be detected using PCR. However, the choice of primers will differ depending upon the method used to generate the model. Transgenic mice should be genotyped across a unique junction fragment. Targeted ES cells used to generate knock-out or knock-in mice should be genotyped using primers from a unique marker in the construct and a region outside of the construct. Targeting in ES cells can also be detected using a genomic Southern blot. Mice targeted using CRISPR/Cas9 should have the region of interest amplified using PCR, and then be assessed for size changes (for large changes in sequence) by Surveyor Assay (for gene knock-out and point mutations) and/or sequenced to verify the mutation. Each of these models has a unique requirement for genotyping, and failure to understand the requirements can easily lead to loss of the gene in subsequent generations.

202 Combination of transcription activator-like effector nucleases and homology-independent target integration strategy gene editing technologies for knock-in of recombinant human factor IX Under the β-casein native promoter in bovine IVF embryos

Precise DNA modification is a crucial approach for gene function elucidation, biomedical model development, and transgenic bioreactor generation. In livestock, its application was extremely challenging until the development of engineered nucleases such as zinc-finger nucleases, transcription activator-like effector nucleases (TALEN), and CRISPR/Cas9. Still, precise knock-in (KI) techniques remain inefficient. Recently, the homology-independent target integration (HITI) strategy was developed, allowing precise insertion of transgenes in mammalian cells in an easier fashion. The HITI technique allows site-specific gene insertion by means of cleavage of both the target sequence in the genome and the donor plasmid, followed by DNA repair by nonhomologous end joining. Here, we evaluated the use of TALENs to generate precise knockout (KO) alleles of the β-casein gene (CSN2) by creating small insertions or deletions, and precise insertion of recombinant human factor IX (rhFIX) under bovine CSN2 regulatory sequences, using HITI via cytoplasmic injection of bovine IVF zygotes. First, 2 TALEN pairs (Tn1 and Tn2) targeting exon 2 of bovine CSN2 were designed and their activity was confirmed by primary fibroblasts transfection followed by Surveyor assay at Day 3. Then, both TALEN pairs were evaluated for KO embryo generation by zygote cytoplasmic injection of in vitro-transcribed mRNA encoding for Tn1, Tn2, or a mix containing Tn1+Tn2, at 100ng μL−1. A non-injected control (NIC) was also included. Embryos were in vitro cultured until Day 7 and independently analysed by whole-genome amplification followed by PCR and sequencing. Neither the blastocyst rate [28.8% (n=73), 33.8% (n=71), 32.4% (n=74), and 54.3% (n=127) for Tn1, Tn2, Tn1+Tn2, and NIC, respectively] nor the proportion of edited embryos [44% (n=9), 20% (n=10), and 33% (n=9) for Tn1, Tn2, and Tn1+Tn2, respectively] differed between injected groups (Fisher test, P&amp;lt;0.05), demonstrating efficient editing in bovine embryos by TALENs. Finally, to achieve precise CSN2 KI embryos, the rhFIX open reading frame was PCR amplified with a forward primer containing the Tn1 recognition sequence to obtain the HITI donor and bovine IVF zygotes were co-injected with the Tn1 mRNA and the HITI donor. Embryos were in vitro cultured until Day 7 and individually analysed by nested PCR at both the 5′ and 3′ ends of HITI donor. The PCR-based results indicate HITI donor integration in 7% of embryos analysed (n=14). Sanger sequencing analysis is currently in progress to confirm site-specific integration of HITI and possible rearranged DNA integration in other embryos. To our knowledge, this is the first report on the use of TALEN and HITI for gene modification. Our results indicate that TALEN combined with HITI may constitute an easy strategy for precise production of pharmaceuticals in the milk of livestock.

Generation of Memory Stem T Cells Specific for Tumor Antigens and Resistant to Inhibitory Signals By Genome Editing

Tumor-infiltrating lymphocytes (TILs) may express different inhibitory receptors (IRs) on the cell surface that are exploited by cancer cells to evade the immune attack. Thus, exhausted T cells display an unresponsive functional profile and fail to kill tumor cells. TIM-3 and LAG-3 represent highly expressed inhibitory receptors in exhausted T cells infiltrating solid tumors. We recently showed that a large fraction of bone marrow infiltrating CD8+ cells co-express multiple inhibitory receptors, including TIM-3, in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplantation. These observations advocate for strategies to generate innovative tumor-specific T cell products resistant to inhibitory signals and able to persist long-term in treated patients. To reach these objectives we developed a strategy to simultaneously redirect T cell specificity by TCR gene transfer and permanently disrupt IRs by CRISPR/Cas9 system in long-living memory stem T cells (TSCM) for adoptive cell therapy.Primary T cells were activated with CD3/CD28-conjugated beads and cultured with low doses of IL7 and IL15, to preserve their TSCM phenotype, and electroporated with Cas9/gRNA ribonucleoproteins (RNPs) targeting a coding sequence of TIM-3,LAG3, 2B4 and of the TCR α and β chain constant region (TRAC and TRBC1/2) genes. Each gene was edited alone or in combination in a multiplex approach. The efficiencies of NHEJ-mediated inactivation of each gene were assessed by cytofluorimetric analysis and confirmed at molecular level by surveyor assay or ddPCR.In order to avoid mispairing between the endogenous TCR and the tumor-specific TCR, we simultaneously inactivated the constant regions of α and β chain of the endogenous TCR genes. Efficiency of TRAC and/or TRBC1/2 gene disruption was 98%, when measured as % of CD3neg cells. TCR knocked-out cells could be efficiently (70-85%) transduced with a lentiviral vector encoding for TCR specific for an HLA-A2 restricted peptide from NY-ESO1.We then focused on IR gene inactivation. Single gene editing produced up to 98%, 93% and 63% genetic knock-out of the TIM-3 , the LAG-3 and the 2B4 genes, respectively. We finally combined one IR inactivation with TCR gene editing in a single protocol. We obtained an average of 68% and 69% of TIM3neg and LAG3neg NY-ESO1-TCR redirected T cells, respectively, and more than 90% of edited cells showed a TSCM phenotype.When tested in functional assays, TCR-IR-edited TSCM cells proved highly effective and specific in killing HLA-A2+ NY-ESO1+ multiple myeloma cells. Of notice, TIM-3neg and LAG-3neg cells proved more effective in producing IFNg, TNFa and CD107a, once challenged with tumor cells.In conclusion, by combining the versatility of multiplex gene editing by CRISPR/Cas9 with culture conditions designed to engineer TSCM cells, we can generate innovative tumor-specific cellular products redirected against tumor antigens and resistant to inhibitory signals. Ongoing in vivo evaluation of tumor-specific IR disrupted memory stem T cells will be reported and discussed. DisclosuresBonini:Intellia Therapeutics: Research Funding.

Construction of EZH2 Knockout Animal Model by CRISPR/Cas9 Technology

背景与目的已有的研究证明CRISPR/Cas9（Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9）系统是一种能够在哺乳动物细胞中高效操作的新型基因编辑技术，应用人工设计的向导RNA（single-guide RNA, sgRNA）介导外源表达的Cas9蛋白与靶点DNA特异性结合以实现对基因组DNA的切割，被切割后的基因组DNA通过非同源重组或同源重组的方式进行修复，从而实现基因的敲除、或者外源基因的敲入等目标。本研究的目的是应用CRISPR/Cas9技术构建小鼠肺部EZH2基因敲除的动物模型。方法针对EZH2基因的编码区，设计两个靶向EZH2基因Exon3和Exon4的sgRNA，通过慢病毒包装、感染细胞、SURVEYOR assay等一系列体外实验，验证所设计的sgRNA的有效性。应用支气管插管的方式把慢病毒灌注到小鼠肺部，利用免疫组化方法和qRT-PCR进行检测。结果NIH-3T3细胞的体外实验结果验证了实验所设计的sgEZH2能够有效地在体外细胞系中介导Cas9切割靶DNA；小鼠支气管插管实验及肺部组织免疫组化和qRT-PCR方法检测EZH2基因敲除的效率，发现实验组小鼠肺部组织EZH2表达明显降低。结论本研究成功设计了两条能够敲除EZH2功能的sgRNA，并应用CRISPR/Cas9技术成功建立了肺部EZH2基因敲除的小鼠模型，为研究EZH2的功能和作用机制提供了有效的动物模型。

High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing.

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

Simultaneous Knockout of CXCR4 and CCR5 Genes in CD4+ T Cells via CRISPR/Cas9 Confers Resistance to Both X4- and R5-Tropic Human Immunodeficiency Virus Type 1 Infection.

Previous research has proven that disruption of either the CCR5 or the CXCR4 gene confers resistance to R5-tropic or X4-tropic human immunodeficiency virus type 1 (HIV-1) infection, respectively. However, the urgent need to ablate both of the co-receptors in individual post-thymic CD4+ T cells for dual protection remains. This study ablated the CCR5 and CXCR4 genes in human CD4+ cell lines and primary CD4+ T cells simultaneously using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a well-developed, highly efficient genetic engineering tool. The efficiency of gene modification is as high as 55% for CCR5 and 36% for CXCR4 in CD4+ cell lines through infection of a single lentiviral vector (LV-X4R5), which were markedly protected from both HIV-1NL4-3 (X4-using strain) and HIV-1YU-2 (R5-using strain) infection. Importantly, approximately 9% of the modified GHOST (3) CXCR4+CCR5+ cells harbor four bi-allelic gene disruptions in both the CXCR4 and CCR5 loci. Moreover, co-delivery of two single-guide RNAs loaded with Cas9: ribonucleoprotein (sgX4&R5 Cas9RNP) disrupted >12% of CCR5 and 10% of CXCR4 in primary human CD4+ T cells, which were rendered resistant to HIV-1NL4-3 and HIV-1YU-2 in vitro. Further, the modified cells do not show discernible mutagenesis in top-ranked off-target genes by the Surveyor assay and Sanger sequencing analysis. The results demonstrate the safety and efficacy of CRISPR/Cas9 in multiplex gene modification on peripherally circulating CD4+ T cells, which may promote a functional cure for HIV-1 infection.

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system

The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was detected. The total number of genetic modifications (29) was higher than the total number of gene-edited embryos, as three blastocysts from the group RNA2X reported more than one type of modification. The modifications included indels (10/56; 17.9%) and large deletions (19/56; 33.9%). Moreover, it was possible to detect HR in 1/8 (12.5%) embryos treated with RNA2X. These results report that the CRISPR/Cas9 system can be applied for site-specific edition of the bovine genome, which could have a great impact on the development of large animals resistant to important zoonotic diseases.

139. Development of a Therapy for Duchenne Muscular Dystrophy Using Either TALEN of Cas9 Proteins

Duchenne muscular dystrophy (DMD) is a hereditary disease due to a mutation of the DMD gene located in the X chromosome. It is mainly caused by the deletion of one or more exons of the DMD gene, which changes the reading frame and results in a premature stop codon and a truncated and inactive dystrophin protein. Nowadays, genome editing by programmable endonucleases like TALENs or the CRISPR/Cas9 systems is a powerful method for developing a treatment for this type of disease. However, the use of DNA encoding these systems leads to a prolonged expression, which may increase the off-target activity of these nucleases. Despite the risk of integration into the genome hence increased probability of side effects, viral vectors remain the most effective delivery system for the nucleases and the sgRNA. The objective of this work was to develop a novel approach using purified TALEN proteins or Cas9 complex (Cas9 protein with crRNA and tracrRNA) for genome editing as potential treatment for DMD. TALEN proteins or Cas9 complex were transduced to generate double strand breaks (DSBs) in the dystrophin gene. Repair of this DSB by non-homologous end joining (NHEJ) could restore the normal reading frame of the DMD gene producing a functional dystrophin protein as already described for Becker patients (Koenig et al. 1989). To develop this approach, DNA coding for a pair of TALEN proteins targeting exon 54 of the DMD gene were engineered and cloned in the pet16b expression vector. The resulting TALEN proteins contained a His-Tag for purification purposes. The proteins were produced in bacteria and the TALEN-His-Tag proteins were purified using a Nickel column. The purity was analyzed by SDS-PAGE electrophoresis. The Cas9 protein, the crRNA and the tracrRNA were obtained commercially. To verify the cleavage activity of the programmable nuclease-complexes, the target genomic region including exon 54 was PCR amplified and the resulting amplicon was incubated with either the TALEN proteins or the Cas9 complex. Cleavage products were analysed by gel electrophoresis. Both types of proteins were able to cut 100% of the amplicons in vitro. The TALEN proteins or the Cas9 complex were tested in Hela IR8 and in human myoblast cell lines. The TALEN proteins were transduced with Cys-(Npys)-(d-Arg)9, Bioporter or the iTop delivery system (propanebetaine). The Cas9 complex was transduced with RNAiMax. The presence of DSBs was evaluated using the Surveyor assay. The Cas9 complex generated the expected cleavage products. These results are similar to those obtained when the plasmids coding for both systems were transfected. This indicates that the Cas9 complex could be used effectively to target a specific gene. In contrary, the TALEN proteins did not induce INDELs detectable by the surveyor assay. This could be due to an insufficient protein transduction. In conclusion, protein therapy using the CRISPR/Cas9 complex could be a promising approach to develop alternative treatments for genetic diseases. We are currently validating this approach in vivo using the hDMD mouse model containing the complete human DMD gene. (‘t Hoen et al. 2008).

325. Targeted Gene Addition Strategies for the Treatment of X-Linked Lymphoproliferative Disease

X-linked lymphoproliferative disease is an immunodeficiency arising from mutations in the SH2D1A gene encoding SAP, a key regulator of immune function expressed in T cells, natural killer, and natural killer T cells. Haemopoietic stem-cell transplantation is curative, and we have shown proof of concept of haemopoietic stem-cell gene therapy in a mouse model. Preliminary data suggests that adoptive transfer of T cells corrected using lentiviral gene therapy can correct immune defects. However, targeted gene editing of T cells may allow for physiologically regulated SAP expression. We constructed SAP-specific TALE-effector nucleases (TALENs) capable of generating a site-specific double-stranded break close to the translation start codon, allowing correction of most described SH2D1A mutations. TALEN activity was validated with a surveyor assay in HEK293T cells. To facilitate optimisation of delivery methods, we designed a fluorescent reporter assay that, when stably integrated into a cellular DNA, provides a fluorescence signal when repair of DNA double-stranded breaks (DSB) created by TALENs occurs. Using the reporter assay in Jurkat cells we observed reduced toxicity and highly efficient delivery using an mRNA nucleofection platform, with 23% of treated cells showing repair mediated fluorescence. We therefore proceeded to deliver TALEN mRNA to human primary T cells, and demonstrated the generation of double-stranded breaks in 25% of the bulk population. To harness the repair of DSB, we constructed a homologous recombination donor in a non-integrating lentiviral vector format to mediate delivery of cDNA with low toxicity. Combined with TALEN mRNA nucleofection, homologous recombination-driven incorporation of donor in T cells was possible. This methodology could be applied to an autologous T-cell gene therapy strategy, although the low level of homologous recombination remains a limiting factor. We plan to test our strategy in patient cell lines to restore SAP expression and cytotoxic function of cytotoxic T lymphocytes.

137. Analysis of Cas9-FokI and TALE-MutH Endonuclease Activity and Toxicity as Key Elements in the Development of a Gene Therapeutic Approach to Treat XLRP

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause severe X-linked retinitis pigmentosa (XLRP). More than 80% of the mutations are located in the terminal exon ORF15 of the RPGR gene. Genome editing, which represents a novel approach to treat monogenic disorders, is based on highly specific nucleases that cleave or nick at a chosen position within the complex genome followed by the repair of the double or single strand break (DSB or SSB) by endogenous repair mechanisms. The major pathways include error prone non-homologous end joining (NHEJ), microhomology-mediated end-joining (MMEJ), and homologous recombination (HR), the latter two with the help of a donor template. Currently, endonucleases for inducing the DSB are based on the CRISPR-Cas9 system or TALE proteins fused to the non-specific FokI nuclease (TALEN). However, specificity and toxicity of both endonuclease types raise concerns about their use for therapeutic in vivo applications. In order to study in vivo genome editing for the treatment of XLRP, our lab has generated a mouse model containing a point mutation in the ORF15 exon. In the present study, we characterize advanced variants of both endonuclease types (Cas9-FokI and TALE-MutH) for their activity and toxicity at the murine Rpgr-ORF15 locus for later usage in the mouse model. In total, ten sequences within or near the ORF15 exon have been targeted for the induction of DSB or SSB. Nine target sites for CRISPR/Cas9-FokI were chosen: three before, within, and behind the exon, respectively, and one target site for TALE-MutH within the exon. These sequences have been cloned into the traffic light reporter (TLR) gene expression system at the homing endonuclease I-SceI site. The TLR system has been modified to express either GFP in case of successful HR or BFP in case of NHEJ. Plasmids containing substrate, nucleases and template DNA were transfected into HEK293T cells. Efficiency of DNA modification was measured by FACS analysis and T7 surveyor assay, and toxicity was assessed by cell survival assay. In addition to the episomal TLR system within a human cell line (HEK293T), the genome of murine C2C12 cells was targeted by all endonuclease variants and toxicity was analysed via the T7 surveyor assay. Toxicity of Cas9-FokI and TALE-MutH are comparable to the golden standard ISceI while standard Cas9 nucleases showed slightly increased toxicity in HEK293T cells. Two different concentrations of the nucleases were used in a toxicity assay and were equally tolerated. Cas9-FokI showed preferences in its activity at the nine target sites with activities well above ISce-I level, while the one target site of TALE-MutH was as efficient as ISceI. Activity results were confirmed in the murine cell line C2C12. Off target toxicity in C2C12 cells was non-detectable. The characterization of the activity and toxicity of the tested endonucleases helped us to identify the most promising tailored nuclease and its target sequence in our gene targeting approach to treat XLRP. With the help of mouse retinal explants and subsequently in vivo experiments, we will confirm the efficacy of endonuclease mediated genome editing in photoreceptors.

Digital detection of endonuclease mediated gene disruption in the HIV provirus.

Genome editing by designer nucleases is a rapidly evolving technology utilized in a highly diverse set of research fields. Among all fields, the T7 endonuclease mismatch cleavage assay, or Surveyor assay, is the most commonly used tool to assess genomic editing by designer nucleases. This assay, while relatively easy to perform, provides only a semi-quantitative measure of mutation efficiency that lacks sensitivity and accuracy. We demonstrate a simple droplet digital PCR assay that quickly quantitates a range of indel mutations with detection as low as 0.02% mutant in a wild type background and precision (≤6%CV) and accuracy superior to either mismatch cleavage assay or clonal sequencing when compared to next-generation sequencing. The precision and simplicity of this assay will facilitate comparison of gene editing approaches and their optimization, accelerating progress in this rapidly-moving field.

Targeted gene addition strategies for the treatment of X-linked lymphoproliferative disease

BackgroundX-linked lymphoproliferative disease is an immunodeficiency arising from mutations in the SH2D1A gene encoding SAP, a key regulator of immune function expressed in T cells, natural killer cells, and natural killer T cells. Haemopoietic stem-cell transplantation is curative, and we have shown proof of concept of haemopoietic stem-cell gene therapy in a mouse model. Preliminary data suggest that adoptive transfer of gene-corrected T cells can correct immune defects. Using gene editing techniques, we aimed to correct the SH2D1A locus in situ allowing physiologically regulated SAP expression. MethodsWe constructed SAP-specific TALE-effector nucleases (TALENs) capable of generating a site-specific double-stranded break close to the translation start codon, allowing correction of most described SAP mutations. TALENs were validated with a surveyor assay in HEK293T cells. To facilitate optimisation of delivery methods, we designed a fluorescent reporter assay that, when stably integrated into a cellular DNA, provides a fluorescence signal when TALEN-mediated DNA double-stranded break repair occurs. FindingsThe ability of TALENs to cleave DNA at the SH2D1A locus was validated with both surveyor and fluorescent reporter assays in HEK293T cells by transfection of plasmid DNA. Using the reporter assay in Jurkat cells we observed reduced toxicity and efficient nuclease activity in an mRNA nucleofection delivery platform. We therefore proceeded to deliver TALEN mRNA to human primary T cells, and demonstrated the generation of double-stranded breaks. We constructed a homologous recombination donor in a non-integrating lentiviral vector format to mediate delivery of corrective cDNA with low toxicity. Combined with TALEN mRNA nucleofection, homologous recombination-driven incorporation of donor in T cells was possible. InterpretationOur data suggest that SAP-specific TALENs can efficiently target the SH2D1A locus and induce double-stranded breaks, which can be used to incorporate donor template SH2D1A cDNA. Targeted gene editing allows us to harness a greater amount of genomic regulatory elements and therefore offers an attractive approach to treat X-linked lymphoproliferative disease. This methodology could be applied to an autologous T cell gene therapy strategy, although the low level of homologous recombination remains a limiting factor. We plan to test our strategy in patient cell lines to restore SAP expression and cytotoxic function of cytotoxic T lymphocytes. FundingWellcome Trust, Academy of Medical Sciences.