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Cpf1 Synthesis

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CRISPR has seemingly endless application possibilities and can be used to make more than double‐strand breaks near a 5′‐NGG‐3′ PAM. Researchers have some reservations regarding the Cas9 enzyme for therapeutic usage because it cannot always make precise edits without making unintended and potential lethal cuts. In 2013 Cho worked to overcome this hurdle in human cells. The strategy Cho used to enhance Cas9 specificity was to use two different Cas9 enzymes that each cut a single stand of the DNA. This is possible because Cas9 has two catalytic domains for cleavage of each strand. Through the use of a single amino acid mutation the catalysid domain is inactivated and the only one of DNA strands is cut. This variation in Cas9 has been termed …show more content…
Zetsche identified a very promising enzyme Cpf1 which is a putative class 2 CRISPR effector. Cpf1 lacks reacerRNA and only requires a crRNA which could similfy the editing process. Cpf1 is also a single RNA-guided endonuclease that uses a T-rich protospacer adjacent motif (PAM). Unlike the blunt end from a Cas9 cleavage, Cpf1 is able to cut DNA through a staggered DNA double strand break with a 5’ overhang that can be used to insert a desired sequence into the genome. They were able to identify 2 enzymes in the Cpf1 protein family that have efficient genome editing activity in human cells (Zetsche, …show more content…
Primarily, it is able to create double stranded cleavages at a specific site based on guide RNA. A second valuable feature that is also being studied within the world of molecular biology is Cas9’s ability to specifically locate a protein of interest. If the Cas9 catalytic domains are mutated the resulting protein call dCas9 is able to locate a specific site in the genome through the sgRNA but not cut the DNA. When researchers attach proteins to the dCas9 they are able to tie specific molecular activities to areas of interest within the genome. In an experiment using dCas9 researchers were able to demonstrate that fusing dCas9 with effector domains allows for stable and efficient transcriptional activation or deactivation in yeast and human cells, at a location determine by sgRNA. When researchers joined dCas9 with a transcriptional repressor domains they were able to strongly silence expression of many genes. Ultimately, researchers were able to conclude through RNA sequence analysis, that CRISPR interference transcriptional repression can allow researchers to precisely regulate gene expression within eukaryotes (Gilbert,

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