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"I’ve been working with iTL over the past 5 years in the production of 3 different genetically altered mice. Not only did iTL help in the design of the mice, […]” - Raghu Mirmira, MD, PhD University of Chicago.
If you’ve read about some of the news regarding breakthroughs in molecular biology and genetics in recent years, you’ve probably already heard about CRISPR Cas9. This revolutionary new tool has allowed scientists to reach new levels of accuracy when it comes to making precise and targeted changes to the genome of living cells for research purposes. Whether this means taking animal testing to a new level or ensuring that stem cell research actually has a future, many goals were already reached with the help of CRISPR/Cas9, and many other – far bolder ones – have been set for the near future.
Following the research of Yoshizumi Ishino and his colleagues at the Osaka University in Japan, in 1987, the accidental cloning of what would later be known as a CRISPR, resulted in a line of research that culminated with the study of interrupted, direct repeats (DR) in organisms like Haloarcula and Haloferax by Francisco Mojica at the University of Alcante in Spain. Even though Mojica’s research proved to lead to an incorrect hypothesis, a student of Mojica’s discovered further repeats in more than 20 species in the year 2000. These repeats were given the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). The CRISPR Cas9 system was devised later using the Cas9 protein to manipulate nucleotide sequences in the guide RNA, and later, to facilitate cleavage for any target DNA sequence, allowing for simplified genome editing.
Since CRISPR and associated Cas genes are used naturally by certain organisms to eliminate invading genetic material, scientists eventually confirmed this function in 2007, and went further to use CRISPR Cas methods artificially in order to facilitate guided targeted gene alterations. CRISPR Cas9 is the primary tool that was designed for this purpose. To achieve location-specific DNA recognition and cleavage, the Cas9 protein had to be put together with two types of RNA, crRNA and trRNA, which is complementary to the former. Through the resulting guide RNA, it was then possible to program the Cas9 protein to alter DNA in a specifically targeted way.
To date, CRISPR Cas9 has become one of the most well-known and frequently adopted genome editing methods used in molecular biology. Different variants of the Cas9 system have been developed during the past decade, and genome-editing protocols were established that would later include systems such as wild-type Cas9, which is responsible for achieving site-specific cleavage in double stranded DNA. Further advancements made possible by researcher Le Cong and his colleagues, taking the Cas9 system to a higher level of efficiency and developing the mutant form Cas9D10A, specifically designed for one-stranded DNA.
The CRISPR Cas9 system has many uses and advantages that have already been recognized by the scientific community. It has already been used successfully for applications such as genome engineering with Cas9 nuclease, genome engineering through the use of double nicking methods, and localization through defective Cas9 nuclease. As for targeting efficiency, Cas9 has scored significantly high, even when compared to other well-known and popular gene editing technologies such as ZFNs or TALENs. Finally, the remarkable thing about the simplified Cas9 system is that it can and has been used successfully for a wide range of medicine and crop seed enhancement applications.
The future of CRISPR/Cas9 is extremely bright. Scientists are already pointing out the rapid advancements that have been made through the use of the technique, and showing that its simplicity has the potential of making it a versatile candidate for a whole range of future projects and assessments. Ultimately, in terms of its use for higher precision DNA cleavage, as well as genome editing and engineering, most experts will admit that the possibilities opened up by CRISPR Cas9 can only be limited by our ability to imagine new uses in molecular biology and genetics.
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