CRISPR is actually a naturally-occurring, ancient defense mechanism found in a wide range of bacteria. As far as back the 1980s, scientists observed a strange pattern in some bacterial genomes. One DNA sequence would be repeated over and over again, with unique sequences in between the repeats. They called this odd configuration as Clustered Regulary Interspaced Short Palindromic Repeats (CRISPR). Originally in 1970s while studying several archae and eubacteria, the scientists came ac-cross such repeats which they called as Short Regularly Spaced Repeats (SRSR). Later in 2000s the clustered nature of these sequences was revealed and thus being named as CRISPR (read as crisper). The further studies revealed that these sequences play a major role to defend bacteria from any phage infection, i.e. in bacterial adaptive immunity.
As I have mentioned earlier, these are short stretches of DNA sequences in bacterial genomes, wherein the bacteria have already encountered any phage invasion in earlier time. These sequences are short repeats mostly 20-50 bp in size and are separated by a 'spacer sequence' which is an artifact of previous phage invasion, i.e. the spacer sequence is a part of phage DNA sequence thus end up protecting bacteria from any subsequent phage invasion.
Such a dyad sequence-structure of the CRISPR loci, leads to formation of hairpin loop like secondary structure although the entire sequence might not be completely palindromic. These repeats are separated by uniform length spacers. In most of the cases the spacer sequences are much identical to the phage DNA sequences. In certain cases the spacer sequence can be identical to prokaryotic DNA sequences making them self targetting.
The CRISPRs are just one part of this bacterial immune system. The other significant counterpart is the CRISPR-associated genes (Cas genes) these genes code for several nucleases. These Cas proteins owing to their nuclease activity can slice any Nucleic Acid sequence. But now the question comes is, what is so special about this system?
So coming to Cas9, Cas9 is widely used nuclease and famous among scientists (albeit notorious amongst the phages ;-) ) and was isolated first from Spy bacteria or full name Streptococcus pyogenes. Now that we know what is the native role of CRISPR/Cas9 system, lets just think for what wonders it may do.
Connecting the dots:
Cas9 is a nuclease which cuts/snips the DNA whereas the CRISPR is a set of sequences which guide the Cas9 as of where to exactly snip the DNA. So since DNA is quite a long stretch of nucleotides A, T, G, C the CRISPR has to guide the Cas9 to some specific 20bp sequence (Cas9 identifies a stretch of 20bp sequence). So all that we biologists do is we study the part of DNA sequence which we want to snip/effiencently engineer, and design a "CRISPR guide RNA" which has the 20bp sequence similar to the target sequence and a tracer RNA sequence for recruitment and snipping purpose os Cas9 protein. SO all one needs to do is design this CRISPR guide RNA sequence and order it cloned inside a suitable vector (usually a plasmid). Upon transfection in to the cells the CRISPR guide RNA and Cas9 shall do their work quite efficiently. Alternatively you can also design a corrected sequence of certain faulty gene (usually in oligo-nucleotide form) and co-transfect it into suitable organism/cell lines harbouring the faulty gene of internest. This shall lead to removal of faulty part from the gene and insertion of the correct part.
Similarly this system can be also used to effectively generate mutant varieties of particular gene for biological experiments. The traditional processes fail to do so with efficiency and require mostly a lot of breeding and may even cost several mice their lives and is time consuming too. The CRISPR system can be used efficiently to mutate any gene of interest and that too with good reproducibilty. Moreover this system can be used to manipulate any organism genome including that of humans.
The only con behind this technique is the off-targetting, that is the Cas9 might also snip a 20bp region which matches the Guide RNA sequence but is not somewhere else in the genome. Various people have been trying to optimise this artifact of off-targets by several computation approaches as well as using different Cas variants of Cpf variants. I wont go there since it is entirely different question to address.
You can refer to this link which tells one of the succesful human treatments by the CRISPR/Cas9 system - A Cell Therapy Untested in Humans Saves a Baby With Cancer
No comments:
Post a Comment