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Posts Tagged ‘CRISPR/Cpf1’


New CRISPR-non Cas9 proteins

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

More CRISPR Proteins Discovered

Researchers identify three new proteins that may serve as alternatives to Cas9.

By Jef Akst | October 23, 2015

http://www.the-scientist.com/?articles.view/articleNo/44323/title/More-CRISPR-Proteins-Discovered/

http://www.the-scientist.com/images/Nutshell/Sept2015/Cas9.jpg

Crystal structure of a Cas9 in complex with an RNA guide and a stretch of target DNAWIKIMEDIA, H. NISHIMASU ET AL.

Scouring genomic databases for sequences with similarity to the components of the CRISPR/Cas9 system and the recently identified CRISPR/Cpf1 system, researchers from the National Center for Biotechnology Information (NCBI), MIT, and Rutgers University have discovered three novel CRISPR systems that could one day provide new gene-editing tools to supplement the currently used CRISPR/Cas9 system. The newly discovered CRISPR systems contain three new proteins, C2c1, C2c2, and C2c3 (named for “Class 2 candidate x”), one of which may cleave RNA.

“This work shows a path to discovery of novel CRISPR/Cas systems with diverse properties, which are demonstrated here in direct experiments,” coauthor Eugene Koonin of NCBI told GenomeWeb. “The most remarkable aspect of the story is how evolution has achieved a broad repertoire of biological activities, a feat we can take advantage of for new genome manipulation tools.” The group published its results yesterday (October 22) in Molecular Cell.

Using such sequence-based techniques, the researchers predict that there are even more CRISPR systems to be discovered, added study coauthor Konstantin Severinov of Rutgers. “There are multiple ways to modify the search algorithm. So more exciting and distinct CRISPR/Cas mechanisms should be expected soon.”

 

Using CRISPR as a High-Throughput Cancer Screening and Modeling Tool

http://www.genengnews.com/gen-news-highlights/using-crispr-as-a-high-throughput-cancer-screening-and-modeling-tool/81251901/

http://www.genengnews.com/Media/images/GENHighlight/thumb_97219_large9423718917.jpg

 

Using CRISPR/Cas9, scientists created a new high-throughput screening tool for studying the development and progression of liver cancer in mice. [Ernesto del Aguila III, NHGRI]
  • A contingent of researchers from the UK, Germany, and Spain have recently developed a novel CRISPR/Cas9 system that they believe can be utilized as a multiplexed screening approach to study and model cancer development in mice. In the current study, the investigators directly mutated genes within adult mouse livers to elucidate their role in cancer development and progression—simultaneously uncovering the gene combinations that coordinate to cause liver cancer.
  • “We reasoned that, by targeting mutations directly to adult liver cells using CRISPR/Cas9, we could better study and understand the biology of this important cancer,” explained co-author Mathias Friedrich, Ph.D., research scientist at the Wellcome Trust Sanger Institute. “Other approaches to engineer mutations in mice, such as stem cell manipulation, are limited by the laborious process, the long time frames and large numbers of animals needed. And, our method better mimics important aspects of human cancer biology than many “classic” mouse models: as in most human cancers, the mutations occur in the adult and only affect a few cells”.
  • The findings from this study were published online recently in PNAS through an article entitled “CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice.”
  • This new approach is rapid, scalable, and extremely efficient, allowing the researchers to examine an array of genes or large regions of the genome concurrently. Moreover, this methodology affords scientists the ability to distinguish between cancer driver mutations and passenger mutations—those that occur as side-effects of cancer development.
  • The research team developed a list of up to eighteen genes with known or unknown evidence for their importance in two forms of liver cancer. They then introduced the CRISPR/Cas9 molecules, targeting various combinations of these genes into mice, which subsequently developed liver or bile duct cancer within a few months.
  • “Our approach enables us to simultaneously target multiple putative genes in individual cells,” noted co-author Roland Rad, Ph.D., project leader at the Technical University of Munich and the German Cancer Research Center Heidelberg. “We can now rapidly and efficiently screen which genes are cancer-causing and which ones are not. And, we can study how genes work together to cause cancers—a crucial piece of the puzzle we must solve to understand and tackle the disease.”
  • The investigators were able to confirm that a set of DNA-binding proteins called ARID (AT-rich interactive domain), influence the organization of chromosomes and are important for liver cancer development. Furthermore, mutations in a second protein, TET2, were found to be causative in bile duct cancer: although TET2 has not been found to be mutated in human biliary cancers, the proteins that it interacts with have been, showing that the CRISPR/Cas9 method can identify human cancer genes that are not mutated, but whose function is disturbed by other events.
  • “The new tools of targeting genes in combination and inducing insertions or deletions in chromosomes change our ability to identify new cancer-causing genes and to understand their role in cancer,” stated senior group leader and co-author Allan Bradley, Ph.D., director emeritus from the Sanger Institute. “Our results show that this approach is feasible and productive in liver cancer; we will now continue to study our new findings and try to extend the approach to other cancer types.”
  • This CRISPR/Cas9 approach may also be favorable for an in-depth examination of genomic deserts —regions within the human genome that appear to be devoid of genes. Yet, recent data from the ENCODE Project suggests that deserts can be populated, if not by genes, then by DNA regulatory regions that influence the activity of genes.
  • “Liver cancer has many DNA alterations in regions lacking genes: we don’t know which of these might be important for the disease,” said Dr. Rad. “However, we could show that it is now possible to delete such regions to systematically determine their role in liver cancer development.”

 

Additional CRISPR Enzymes Found Bioinformatically

http://www.genengnews.com/gen-news-highlights/additional-crispr-enzymes-found-bioinformatically/81251889/

  • Three newly discovered enzymes—provisionally named C2c1, C2c3, and C2c3—promise to expand the CRISPR genome-editing toolbox beyond the well-known Cas9. They had been hiding inside NIH genomic databases, but they were eventually found out, thanks to the application of computational approaches developed by two groups of researchers. These researchers also initiated experimental work to explore the function of the bioinformatically identified enzymes.

One of the groups was led by Eugene Koonin, Ph.D., senior investigator at the National Center for Biotechnology Information (NCBI), National Library of Medicine (NLM), part of the NIH. The other group was led by Feng Zhang, Ph.D., of the MIT-Harvard Broad Institute.

According to the researchers, the three newly discovered enzymes are all naturally occurring, and all share some features with Cas9. In addition, these three enzymes have unique properties that could be exploited for novel genome-editing applications.

“This work shows a path to discovery of novel CRISPR-Cas systems with diverse properties, which are demonstrated here in direct experiments,” said Dr. Koonin. “The most remarkable aspect of the story is how evolution has achieved a broad repertoire of biological activities, a feat we can take advantage of for new genome-manipulation tools.”

This comment highlights how the researchers’ work, which appeared October 22 in the journal Molecular Cell, included information about potential evolutionary pathways. The researchers also emphasized that their work might lead to additional enzyme discoveries.

“There are multiple ways to modify the search algorithm, so more exciting and distinct CRISPR-Cas mechanisms should be expected soon,” said Konstantin Severinov, Ph.D., one of the researchers. He is affiliated with Rutgers and the Skolkovo Institute of Science and Technology. “These new mechanisms will undoubtedly attract the attention of basic and applied scientists alike.”

The Koonin and Zhang groups also recently collaborated on a project that resulted in the characterization of Cpf1, a class II CRISPR endonuclease, like Cas9. This work was described last month in an article, published in Cell, suggesting that the newly found enzyme’s distinct features pointed to unique genome-editing possibilities.

In his comments about this earlier work, Dr. Zhang made a point that presaged the current work: “Our goal is to develop tools that can accelerate research and eventually lead to new therapeutic applications. We see much more to come, even beyond Cpf1 and Cas9, with other enzymes that may be repurposed for further genome-editing advances.”

 

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