분자유전학실험실 (단국대학교 분자생물학과)



 이성욱 ( 2016-08-30 08:18:05 , Hit : 1418
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 DNA Can Be Edited Without Being Cut




http://www.genengnews.com/gen-news-highlights/dna-can-be-edited-without-being-cut/81253144/


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Aug 29, 2016


DNA Can Be Edited Without Being Cut
Deaminase is attached by a linker to nuclease-deficient CRISPR/Cas9. Guide RNA recognizes the DNA sequence of target genome and the deaminase modifies the base of the unwound DNA. [Kobe University]

  

The figure analyzes the frequency at which mutation was induced in 40 bases of the target DNA sequence. In the nuclease model (left), mainly insertion and deletion are induced. In the deaminase model (right), only point mutation is induced. [Kobe University]


Scientists at Kobe University in Japan have developed a new gene-editing technique by combining elements of a bacterial immune system, CRISPR/Cas9, and a vertebrate immune system, AID, or activation-induced cytidine deaminase. The new technique, called Target-AID, preserves the CRISPR/Cas9’s DNA-targeting machinery but dispenses with its DNA-cleaving functionality. In place of DNA cleavage, Target-AID employs the base-altering functionality of AID.

According to the Kobe University scientists, Target-AID may be free of one of the complications associated with the CRISPR/Cas9, namely, the risk of cytotoxicity. Cytotoxicity may occur if the CRISPR/Cas9 system’s nuclease activity results in chromosome splitting.

The Kobe University team anticipates that Target-AID will be applied to gene therapy in the future. More immediate applications include the breeding of organisms that will be useful in disease and drug-discovery research.

In bacteria, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) records and cleaves invasive foreign DNA. A ribonucleoprotein complex of Cas nuclease and guide-RNA (gRNA) binds to complementary target DNA sequences, forms an R-loop, and then cleaves the DNA. In vertebrates, AID is responsible for targeted hypermutation by modifying the deoxycytidine of the variable region of the immunoglobulin locus.

AID creates mutations in DNA by deaminating cytosine, thereby turning it into uracil. Essentially, AID changes a C:G base pair into a U:G mismatch. Afterward, when the cell's DNA replication machinery recognizes the U as a T, the C:G is converted to a T:A base pair. Such base-pair changes ensure the production of diverse secondary antibodies through a process known as somatic hypermutation.

Target-AID, the Kobe University scientists found, succeeded in modifying genetic function by inducing target point mutations at a highly efficient rate. The scientists also observed that the toxicity associated with the CRISPR/Cas9 system was greatly reduced.

These findings appeared August 4 in the journal Science, in an article entitled, “Targeted Nucleotide Editing Using Hybrid Prokaryotic and Vertebrate Adaptive Immune Systems.” The article’s authors included senior author Akihiko Kondo, Ph.D., and first author Keiji Nishida, Ph.D., both of Kobe’s Graduate School of Science, Technology, and Innovation.

“Nuclease-deficient type II CRISPR/Cas9 and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis,” wrote the article’s authors. “Specific point mutation was induced majorly at cytidines within the target range of five bases.

The Kobe scientists noted that their hybrid approach could be extended by incorporating different cytidine deaminases. For example, the cytidine deaminase used in the current study, PmCDA1, is derived from the sea lamprey. It could be replaced by another cytidine deaminase, rAPOBEC1, which is a rat apolipoprotein B mRNA editing enzyme catalytic polypeptide.

“Although direct comparison will be needed, these two systems may complement each other to extend the repertoire of possible editing sites,” explained the authors of the Science article. “Using other CRISPR-related systems or other modifier enzymes such as adenosine deaminase also will broaden the editing capacity and further enrich the genome editing toolbox.”

The authors indicated that the combination of nickase Cas9(D10A) and the deaminase induced insertion and deletion (indel) in mammalian cells. However, use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the system’s efficiency.







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