Cas9 And CRISPR As Novel Molecular Genetics Tool...#biopharma #medtech #CRISPR
Biology of cas 9:
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes functions are necessary in adaptive immunity in bacteria and archea that enable the organisms to respond and remove invading genetic stuff. These repeats were discovered in the 1980s in E. coli, but their function was not confirmed until 2007. Barrangou and colleagues demonstrated that S. thermophilus could attain resistance against a bacteriophage via integrating a genome fragment of the infectious virus into its CRISPR locus.
Invading DNA from viruses or plasmids is initially cut into tiny fragments and incorporated into a CRISPR locus among a series of short repeats. The loci are then transcribed, and transcripts are processed to create small RNAs, which are then used to guide effector endonucleases that target foreign DNA based on sequence complementarity.
Cas9 processes crRNAs and causes the destruction of the target DNA. Cas9’s function in both steps relies on the presence of the two-nuclease domains, a RuvC-like nuclease domain that is located at the amino terminus and a HNH-like nuclease domain residing in the mid-region of protein.
Cas9 must form complex with a crRNA and a trans-activating crRNA to get site-specific DNA cleavage as well as recognition that is partially complementary to the crRNA. The tracrRNA is required for crRNA maturation from a primary transcript that encodes multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9.
During the destruction of invading DNA, the HNH and RuvC-like nuclease domains nick the both DNA strands, which generate double-stranded breaks (DSBs) at sites characterized by a 20-nucleotide target sequence present within an associated crRNA transcript. The HNH domain breaks the complementary strand; on the other hand, RuvC domain cleaves the non-complementary strand.
The double-stranded endonuclease activity of Cas9 also demands that a short conserved sequence, (2–5 nts) known as protospacer-associated motif (PAM), proceeds immediately after 3´- of the crRNA complementary sequence. In fact, fully complementary sequences are ignored by Cas9-RNA if PAM sequence is absent.
CRISPR AND CAS 9 AS GENETIC TOOL:
The simplicity of the type II CRISPR nuclease makes it amenable to adaptation for genome editing. The Doudna and Charpentier labs realized this in 2012. Based on the type II CRISPR system simplified two-component system was developed by combining crRNA and trRNA into synthetic single guide RNA (sgRNA). sgRNA programmed Cas9 was as effective as Cas9 programmed with separate crRNA and trRNA in guiding specified gene alterations.
OFF TARGET MUTATIONS AND TARGETING EFFICIENCY
Targeting efficiency is one of the most important parameters to assess a genome-editing tool. The targeting efficiency of Cas9 is favorable as compared to more established methods, such as TALENs. For example the Cas9 system has the efficiencies >70% in plants and zebrafish and ranges from 2–5% in induced pluripotent stem cells. In addition, Zhou and colleagues improves genome targeting up to 78% in one-cell embryos of mouse, and achieved effective germline transmission by exploiting dual sgRNAs to target an individual gene simultaneously.
T7 Endonuclease I mutation detection assay a widely used method to identify mutations is the. This assay detects heteroduplex DNA that is the results of annealing of a DNA strand, having desired mutations, with a wild type DNA strand.
APPLICATIONS AS GENOME TARGETING AND GENOME EDITING TOOL
The CRISPR/Cas9 system has been widely adopted following its initial demonstration in 2012. It has already been used successfully to target important genes in many cell lines and organisms, including human, zebra fish, bacteria, C. elegans, plants, yeast, Drosophila, Xenopus tropicalis, monkeys, rabbits, pigs, mice andrats. Several groups have exploited this method to introduce single point mutations (insertions or deletions) in a target gene by a single gRNA. By using pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce inversions or translocations. A recent development is the use of the dCas9 of the CRISPR/Cas9 system for protein domains targeting for transcriptional regulation, microscopic visualization of specific genome loci and epigenetic modification.
The CRISPR/Cas9 system needs only the redesigning of the crRNA to change target specificity. This is different from other genome editing tools, such as zinc finger and TALENs that requires redesign of the protein-DNA interface. Furthermore, CRISPR/Cas9 allows rapid genome-wide interrogation of gene function by creating large gRNA libraries for genomic screening.
THE FUTURE OF CRISPR & CAS 9
Due to the simplicity, versatility and high efficiency of the system the rapid progress in developing Cas9 into a set of tools for molecular and cell biology research has been remarkable. Among the designer nuclease systems currently available for precise genome engineering, the CRISPR/Cas system is by far the most users friendly. It is now clear that Cas9’s ability reaches beyond DNA cleavage, and its usefulness for locus-specific recruitment of proteins for genome will possibly only be limited by our imagination.