CRISPRs technology has become the latest new darling, and in just one month in March 2013, 5 articles have been published in magazines including Science and Nature Biotechnology Related papers introduce this simple and practical new technology for genome modification, now known as CRISPR–Cas systems.
Recently, researchers from Harvard University, Broad Institute of MIT and Harvard and other institutions used CRISPRs technology to edit the genome of human pluripotent stem cells, and pointed out that this method is more effective than TALENs method. Related research papers were published in the recently published "Cell Stem Cell" magazine.
CRISPRs are called Clustered regularly interspaced short palindromic repeats. This is a type of repeating structure widely distributed in the genomes of bacteria and archaea. Studies have shown that CRISPR, along with a series of related proteins and leader sequences, can provide prokaryotic organisms with acquired immunity against foreign genes such as phages.
In addition, in January 2013, Harvard geneticist George Church and his colleagues used the CRISPR method to successfully edit the genomes of several different human cell lines, including induced pluripotent stem cell iPS. This and another Science article proved for the first time that Cas9 nuclease can indeed be used to edit the genome of mammalian cells.
The researchers pointed out that CRISPR has great advantages compared to other genome engineering technologies, such as zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENS). CRISPR is easier to operate and more scalable. .
In order to further compare the relative efficiency of TALENs and CRISPRs, in this article, the researchers used these two methods to analyze the same genomic locus of the same hPSC cell line using the same platform. The efficiency of TALENs for cloning a mutant allele is 0% -34%, while the efficiency of CRISPRs method is 51% -79%, and the latter can also easily generate homozygous mutant clones (7 % -25%), in addition, this method is also nearly ten times higher than the TALENs method.
The researchers speculate that this outstanding performance of CRISPRs is due to the higher expression of Cas9 protein, which is also better tolerated than TALENs in hPSCs. Other reasons may also be that Cas9's endogenous DNA is not curled Activity, and TALEN will bind to methylated DNA after damage.
However, CRISPRs also have some shortcomings, among which two points are particularly noteworthy: First, the requirements of G (N) 19NGG sometimes become restrictive, and secondly, the off-target rate of the CRISPRs method needs to be further determined.
The mechanism of action of this structure may be similar to the RNA interference process of eukaryotes. It was first discovered in 1987 in the flanking sequence of the iap gene of Escherichia coli K12. In February this year, researchers from Harvard University used the TALEN method in another article "A TALEN genome-editing system for generating human stem cell-based disease models" to quickly generate in cultured somatic cells and human pluripotent stem cells. 15 mutant alleles have been identified, among which pluripotent stem cells can differentiate into various metabolic cell types. This can be used in disease research models.
These analyses further point out the advantages and disadvantages of various aspects of the CRISPRs method. If you need to conduct research in this area, you can read this important document in detail.
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