Nowadays, the application of custom-engineered sequence-specific nucleases enables genetic changes in human cells to be easily made with much greater efficiency and precision, such as CRISPR/Cas9, and TALEN. Engineered double-stranded DNA breaks are able to efficiently disrupt genes, also which can be realized by the right donor vector, engineer point mutations, as well as gene insertions. Nevertheless, in order to ensure the maximum gene targeting efficiency and specificity, a great variety of design considerations should be taken into account. It is especially important when engineering induced pluripotent stem cells (iPSCs), because they are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. With years of exploration in iPSC development, Creative Biolabs is dedicated to providing a full range of iPSC genome editing services for customers all over the world.
Viral vectors are good candidate to be used for transient transfection. Generally, lentiviral vectors are utilized to bring Cas9 and sgRNA components into a great number of cell types, both dividing and non-dividing. Besides, integrase-deficient lentiviral vectors (IDLV) have also been applied to deliver nucleases and homologous donor templates to edit iPSCs, these vectors can deliver gene constructs which do no integrate into the genome and are gradually lost during cell division. Moreover, adenoviral vectors are able to be used to edit human stem cells in vitro, because they can transduce a number of dividing and non-dividing cells.
Xanthomonas is a genus of Proteobacteria, it utilizes the type III secretion system to inject effector proteins into plant cells, in order to overcome and reprogram the cellular machinery of the host. A set of these effector proteins, named transcription activator-like effectors (TALEs), are targeted to the nucleus. TALEs are hybrid proteins comprised of DNA-binding modules from TALE proteins and the FokI catalytic domain for the production of site-specific DSBs. In order to introduce a site-specific DSB into the genome, two TALENs should be engineered to bind to the forward and reverse strands with an optimal spacer sequence to enable dimerization of the FokI domain, leading to the formation of DSBs.
Fig. 1 Part a shows the ZFNs based genome editing approach, Part b shows the TALENs based genome editing approach, Part c shows the Crisprs/Cas systems based genome editing method. (Eid, A., 2016)
Bacteria and archaea fend off invading nucleic acids from phages and conjugative plasmids by using clustered regularly interspaced palindromic repeats Crisprs/Cas systems. The CRISPR adaptive immunity systems function via the orchestrated and cooperative activities of several proteins to target invading nucleic acids, such as DNA or RNA.
CRISPR/Cas systems act as molecular immunity machinery to keep a molecular record of previous invaders through a short spacer sequence. The function of this spacer sequence is targeting and destroying invading nucleic acids (CRISPR RNA or crRNA) in future invasions. There are a variety number of CRISPR/Cas systems which primarily target DNA and or RNA molecules in various ways via multi-ribonucleoprotein complexes. Therefore, this system has been widely used to edit the genomes of a diverse array of mammalian cell types and organisms with great efficiency and precision, including iPSCs.
With our advanced iPSC development platform, we offer high quality custom-built iPSC genome editing services, to help customers reach the following goals.
With professional scientists devoted themselves in iPSC genome editing, Creative Biolabs is dedicated to providing the first class genome editing services of iPSC for our customers. Please contact us for more information and a detailed quote.
As a stem cell biotechnology company, we take immense pride in our iPSC genome editing services. Here are some of the key features:
Every project concludes with rigorous quality control, followed by validation to ensure that the precise genetic alteration was made without off-target effects. We offer full project support from our team of experienced stem cell biologists, ensuring smooth communication and execution of your project requirements.
Apart from the regular service, we offer post-delivery support to help troubleshoot any issues that clients may encounter while working with the engineered iPSCs. These are part of our commitment to providing the best quality services to our clients who are at the forefront of stem cell research and therapeutic development.
Reference
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