A series of techniques have been developed to create targeted and efficient genetic modification in both eukaryotic and mammalian species in recent years, enabling the extensive application of laboratory mice in biomedical research. Among the current generation of genome editing technologies, the most rapidly developing new tool is based on a CRISPR-associated endonuclease, known as Cas9, from the microbial adaptive immune system against invading viruses.
Owing to its simplicity in design and superior efficiency, the CRISPR/Cas9 system has become the technology of choice for genome editing in both cell lines and animal model creation. In particular, Creative Biolabs offers this latest gene editing technique for our clients to custom their desired genetically engineered mouse models.
Technology Background
Before the CRISPR/Cas9 approach was discovered to be a potential gene editing tool, several attempts had been made by researchers over the years to manipulate gene function, including homologous recombination (HR) and transcription-activator-like effector nucleases (TALENs). However, each of these platforms has unique limitations. On one hand, although HR-mediated gene targeting produces highly precise alterations, the desired recombination events occur extremely infrequently (1 in 106-109 cells). On the other hand, although effective genome editing is achieved via nuclease-based methods like TALENs, these approaches are costly and time-consuming to engineer, restricting their widespread use, particularly for large-scale, high-throughput studies.
Mechanisms of CRISPR/Cas system
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are segments of prokaryotic DNA containing short repetitions of base sequences, which are separated by segments of exogenous DNA, called spacers. Specifically, the system comprises a shot guide RNA (crRNA and tracrRNA) and one protein component, Cas9 protein. The crRNA is a short RNA that recognizes the target DNA via Watson-Crick base pairing while the tracrRNA hybridizes with the crRNA. Subsequently, the Cas9 nuclease can interact with the DNA:RNA complexes and cleaves the DNA at a specific site. Following DNA cleavage, the break mainly gets repaired through a mechanism called non-homologous end joining (NHEJ), resulting in insertions and/or deletions (indels) which disrupt the targeted locus due to the highly error-prone characteristics of NHEJ pathway. Such an event, in most cases, results in a frame-shift mutation of the coding sequence, eventually leading to gene knock-out. Alternatively, if a third component, a donor template with homology to the targeted locus is supplied, the double-strand break may be repaired by the homology-directed repair (HDR) pathway allowing for the introduction of specific mutations to be achieved (called knock-in). The donor template DNA can be either a single-stranded oligonucleotide or a double-stranded plasmid/linear DNA.
Features of CRISPR/Cas9 system
The CRISPR/Cas9 Process
At Creative Biolabs, a group of highly experienced experts will be assigned to work with you to design and customize your genetically engineered animal models, including both knock-out and knock-in models. The F1 mice can be obtained as little as 5 months using our CRISPR/-Cas-9 technologies. The process involves five major steps:
Meanwhile, Creative Biolabs offers other related techniques used for gene manipulation in the generation of genetically engineered models that you might be interested in. Please click the links below for more information.
Supported by cutting-edge technologies and seasoned specialists in this field, Creative Biolabs offers the most comprehensive model creation service portfolio in the industry. We are proud to offer the most reliable genome engineering model services at most favorable prices currently on the market. Contact or inquire us for more detailed information or a formal quote.
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