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TARGATT™ for Targeted Gene Insertion in Mouse Models

2016-TARGATTmouse-whitepaper

Applied StemCell Inc.’s proprietary TARGATT™ knock-in mouse technology enables highly efficient site-specific gene integration in mammalian cells and animals. This technology uses PhiC31 integrase to insert any gene of interest into a specific docking site that was pre-engineered into an intergenic and transcriptionally active genomic locus for guaranteed gene expression. TARGATT™ Technology can be utilized for generating mouse models for a variety of applications including reporter gene expression, gene knockdown, disease models and even site-specific Cre expression for advanced conditional knock-out models. We have compiled and reported data from our mouse model projects in a technical white paper titled TARGATT™  FOR TARGETED GENE INSERTION IN MOUSE MODELS. This paper highlights the advantages of this novel gene editing platform in generating site-specific knock-in mouse models, and Applied StemCell's expertise in successfully generating transgenic mice.


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AACR 2017 - Using CRISPR/Cas9 to generate isogenic cell lines and reference standards for applications in cancer diagnostics

2017-AACR-poster

We are entering an era of precision medicine, in which there are significant initiatives to employ companion diagnostics and molecular profiling to inform clinical decisions in therapeutic treatment. In order for these initiatives to work as effectively as possible, there is a need for molecular reference materials that can help facilitate precision assay development. Cell-line based reference standards are ideal for such applications because they represent a reproducible and renewable source of control materials that can be employed for assay development, quality control, or lead compound discovery.  With this in mind, we have used the CRISPR/Cas9 platform to engineer series of isogenic cell lines that feature 51 recurrent mutations in the MAPK signaling pathway. These cell lines contain footprint-free, homozygous mutations in the kinase domains of the EGFR, KRAS, and BRAF genes in HCT116 and RKO cancer cell lines, with 32 mutations in EGFR, 12 mutations in KRAS, and 7 mutations in BRAF. The mutations include precise deletions, point mutations and small fragment insertions, and these engineered cell lines have been further employed for the development of derivative reference materials, including genomic DNA (gDNA) and FFPE cell line blocks.  Such materials can be employed as positive and negative controls in diverse assay platforms, including next generation sequencing (NGS), quantitative PCR (qPCR), in situ hybridization (ISH), and others. We present quality control data for these reference materials, which demonstrate excellent consistency and uniformity, as well as examples of the application of these materials in molecular assay validation.  We conclude that using CRISPR/Cas9 technology to generate molecular reference standards is a very efficient and precise method in generating footprint-free, biorelevant mutations for companion diagnostic assays.

AACR abstracts



2016-dot-poster

Site-specific Gene editing in patient-specific induced Pluripotent Stem Cells (iPSCs) offers one of the most promising approaches for the personalized investigation of the pathophysiology of disease and has great therapeutic potential in regenerative medicine. CRISPR/Cas9 works most efficiently for gene knockout through the non-homologous end joining (NHEJ) pathway, and for point mutation/ correction through homology directed repair (HDR). However, the efficiency for gene knock-in is not as satisfactory. Here, we present a few examples of gene knock-in, knock-out, and point mutation with CRISPR/Cas9 in human iPS cells. We also present an alternative route to efficiently generate large fragment knock-in using a “Docking-site-ready” iPSC master line. The master cell line was generated by inserting an integrase recognition “attP” site into a specific genomic safe harbor locus using CRISPR/Cas9. We show data that with the TARGATT™ iPSC master cell line and integrase system, we can knock-in any transgene (up to 20 kb) at the preselected docking site with high efficiency (up to 30-40%), and without drug selection or cell sorting. The TARGATT™-iPSC cell lines is a very efficient tool for reporter gene knock-in, generating inducible gene expression models and these data support that CRISPR/Cas9 and TARGATT™ together provides a valuable new platform for efficient genome editing in human iPSCs.



2016-merckposter
This poster discusses details about ASC's milestones in editing blood-derived cell lines. The CRISPR/Cas9 technology has revolutionized genome editing in eukaryotic cells and embryos. Here we present our data based on our hands-on experience in blood-derived cell lines: (a) gRNA activity discrepancy in three major blood derived immune cell lines, Jurkat, K562 and TF1 cells; (b) Efficiency evaluation of the cloning-free CRISPR system by testing synthesized gRNAs with Cas9 expression plasmids; (c) Solutions for blood cells that are sensitive to transfection with DNA plasmids, such as the bone marrow derived BCWM.1 cells and the natural killer leukemia cells, KHYG1.
 

ISSCR 2016 – Gene editing and neuronal differentiation in human iPSC

2016-ISSCRposter

Genome editing/correction in patient-specific induced pluripotent stem cells (iPSCs) offers one of the most promising approaches for personalized therapy in regenerative medicine. CRISPR/Cas9 has been widely used as an effective gene editing tool in the past two years for site-specific gene modification including insertion, deletion and nucleotide replacement. CRISPR works most efficiently in gene knockout through the non-homologous end joining (NHEJ) pathway. However for DNA knockin, the efficiency of CRISPR mediated homology-directed repair (HDR) remains low, limiting its application in therapeutics.

Our TARGATTTM technology allows site-specific gene insertion at a higher efficiency in a genomic safe harbor locus. We will discuss the results on knockin, knockout and point mutation efficiency in iPSCs using CRISPR/Cas9. We will also report using a “master” iPSC line for efficient site-specific insertion of large DNA fragments. With TARGATTTM and CRISPR, we are empowered with a complete set of genome editing tools to manipulate human iPSCs. 

ISSCR Poster Abstract - #W2087

World Preclincal Congress 2016 – CRISPR-TARGATT™ gene editing in iPSC

2016-WPC poster

Gene editing in patient-specific induced Pluripotent Stem Cells (iPSCs) offers one of the most promising approaches for personalized therapy in regenerative medicine. CRISPR/Cas9 works most efficiently for gene knockout through the non-homologous end joining (NHEJ) pathway, and for point mutation/ correction through homology directed repair (HDR). However, efficiency for large fragment DNA knock-in through nuclease mediated HDR is very low.  Here, we present a few examples of knock-out, point mutation and large fragment knock-in with CRISPR/Cas9 in human iPS cells. We also present an alternative route to efficiently generate large fragment knock-in using a “Docking-site-ready” iPSC master line. The master cell line was generated by inserting an integrase recognition “attP” site into a specific genomic safe harbor locus using CRISPR/Cas9. We show data that with the TARGATT™ iPSC master cell line and integrase system, we can knock-in any transgene (up to 20 kb) at the preselected docking site with high efficiency (up to 30-40%), and without drug selection or cell sorting. These data support that CRISPR/Cas9 and TARGATT™ together provides a valuable new platform for efficient genome editing in human iPSCs.

WPC - Poster titles

AACR 2016 - CRISPR editing in mouse

2016-AACRposter
 
Applied StemCell is one of the first and most experienced provider of the CRISPR-Cas9 Technology for mouse model generation. Our expert team of scientists have extensively researched and upgraded the CRISPR system to optimize the efficiency of Cas9 cutting, modification efficiency and even improve birth rate of mice. The findings were presented at the prestigious AACR Annual Meeting 2016 in New Orleans in April. Some of key findings have been noted under technical details.

ISTT 2016 - TARGATT™ Rat Models

2016-ISTTposter

The laboratory rat (R. norvegicus) is a central experimental animal in several fields of biomedical research, such as cardiovascular diseases, aging, infectious diseases, autoimmunity, cancer models, transplantation biology, inflammation, cancer risk assessment, industrial toxicology, pharmacology, behavioral and addiction studies, and neurobiology. Until recently, the ability to create genetically modified rats has been limited compared to that in the mouse, mainly due to lack of genetic manipulation tools and technologies in the rat. The isolation and establishment of rat embryonic stem (rES) cells by Drs. Ying and Smith’s groups at the end of 2008 enhanced the capability of making genetically modified rat models. Recent advances in nucleases such as ZFNs, TALENs, and particularly, CRISPR have been successfully used to generate ‘knockout’ rat models by injecting gene targeting molecular complexes directly into an embryo. As a result, rat transgenics is becoming as precise and powerful as has been the case in mice, yielding better models of human diseases.

To facilitate the generation and use of rat models of human diseases, it is critical to develop systems that enable fast, efficient and precise introduction of exogenous genetic elements into the rat genome. We have previously developed an integrase-based TARGATTTM system for making site-specific transgenic mice. Here, we applied the same TARGATTTM approach to direct transgene integration at transcriptionally active locus of the rat genome. Integrases such as phiC31 carry out efficient, unidirectional recombination between two non-identical sites, attP and attB. We first identified a transcriptionally active locus in the rat genome and inserted attP sites at the locus using CRISPR. These attP-containing rats can be used as embryo donors for pronuclear injection of the transgene on an attB-containing plasmid. In the presence of integrase, recombination between attP and attB results in an insertion of the transgene precisely at the attP site in the rat genome. This technology allows fast, efficient generation of knock-in rat models containing any gene of interest with consistent, stable, guaranteed gene expression. Advantages of this integrase-based technology are: (1) Transgene integration happens at pre-selected and transcriptionally active locus; (2) Site-specific knock-in rat models are made by direct injection of the DNA into the rat zygotes, bypassing rES cells; (3) Gene integration efficiency is higher, but off-target events are lower compared to CRISPR. This TARGATTTM rat system offers a cost-effective method and valuable resources for the bio-medical community who employ rat models for their studies of human diseases. 


Tricon 2016 – Gene editing in blood lineage cells

2016-Triconposter

The bacterial CRISPR-Cas9 system has been tested successfully from test tubes to eukaryotic cells, and has emerged as a genome editing for multi-organisms. Here, we focus on examining the CRISPR system and compare its efficiency in blood-derived immune cell lines, Jurkat, K562, and TF1 cells. Eight gRNAs were tested among these 3 lines. We found the activities of the most gRNAs are similar in K562 and Jurkat cells, while seven out of eight gRNAs show significant lower activity in TF1 cells, suggesting that loci eligibility and efficiency of CRISPR are different not only from tissue to tissue but also from cells in the same lineage.

Guide RNA synthesis offers lower cost and efforts comparing to traditional plasmid-based sgRNA cloning approach. To evaluate this cloning free CRISPR system, we tested synthesized gRNAs with Cas9 expression plasmids. We found that when co-transfected with Cas9 DNA plasmids, synthesized gRNAs do not work, while co-transfection of U6-gRNA plasmids with Cas9 plasmid showed high efficiencies; Alternatively,  transfection of these synthetic gRNAs in Cas9 expressing cells also led to effective Cas9 cleavage, suggesting that the synthetic gRNAs possess shorter lifespan and fail to synchronize with the Cas9 expression when using DNA plasmids. 

ISTT 2015 - TARGATT™ Master Cell Line