Breakthrough shows how DNA is 'edited' to correct genetic diseases

May 26, 2014
DNA

An international team of scientists has made a major step forward in our understanding of how enzymes 'edit' genes, paving the way for correcting genetic diseases in patients.

Researchers at the Universities of Bristol, Münster and the Lithuanian Institute of Biotechnology have observed the process by which a class of enzymes called CRISPR – pronounced 'crisper' – bind and alter the structure of DNA.

The results, published in the Proceedings of the National Academy of Sciences (PNAS) today, provide a vital piece of the puzzle if these genome editing tools are ultimately going to be used to correct genetic diseases in humans.

CRISPR enzymes were first discovered in bacteria in the 1980s as an immune defence used by bacteria against invading viruses. Scientists have more recently shown that one type of CRISPR – Cas9 – can be used to edit the - the complete set of genetic information for humans.

These enzymes have been tailored to accurately target a single combination of letters within the three billion base pairs of the DNA molecule. This is the equivalent of correcting a single misspelt word in a 23-volume encyclopaedia.

To find this needle in a haystack, CRISPR enzymes use a molecule of RNA - a nucleic acid similar in structure to DNA. The targeting process requires the CRISPR enzymes to pull apart the DNA strands and insert the RNA to form a sequence-specific structure called an 'R-loop'.

The global team tested the R-loop model using specially modified microscopes in which single DNA molecules are stretched in a magnetic field. By altering the twisting force on the DNA, the researchers could directly monitor R-loop formation events by individual CRISPR enzymes.

This allowed them to reveal previously hidden steps in the process and to probe the influence of the sequence of DNA bases.

Professor Mark Szczelkun, from Bristol University's School of Biochemistry, said: "An important challenge in exploiting these exciting genome editing tools is ensuring that only one specific location in a genome is targeted.

"Our single molecule assays have led to a greater understanding of the influence of DNA sequence on R-loop formation. In the future this will help in the rational re-engineering of CRISPR enzymes to increase their accuracy and minimise off-target effects. This will be vital if we are to ultimately apply these tools to correct in patients. "

Explore further: A CRISPR way to edit DNA

More information: 'Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes' by Mark D. Szczelkuna, Maria S. Tikhomirovab, Tomas Sinkunasd, Giedrius Gasiunasd, Tautvydas Karvelisd, Patrizia Pscherac, Virginijus Siksnysd and Ralf Seidel in PNAS: www.pnas.org/cgi/doi/10.1073/pnas.1402597111

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JVK
1 / 5 (5) May 26, 2014
Given what is known about protein folding that links RNA to cell type differentiation, this report, which is related to the one on the understanding of how enzymes 'edit' genes, seems scary. However, the conserved molecular mechanisms of nutrient-dependent protein folding also link naturally-induced epigenetic changes in plants to microRNAs in our circulation.

http://dx.doi.org.../505621a
alfie_null
2 / 5 (1) May 27, 2014
Editing genes to correct genetic diseases is merely a subset of editing genes for any purpose. And we will do so; the pressure to do it will be irresistible. Fun (or horrifying) to predict the consequences. Even the most subtle, most innocuous traits, especially if selected by large numbers of parents, can (probably will) have a large, unforeseen effect on society some years later as those children grow into adults.
thingumbobesquire
1 / 5 (2) May 27, 2014
So, according to reductionists' schemas DNA is the be all and end all of what makes everything living tick. But what is guiding these tiny enzymes? It must be of a higher quasi intelligent species than DNA. No?
supamark23
5 / 5 (4) May 27, 2014
So, according to reductionists' schemas DNA is the be all and end all of what makes everything living tick. But what is guiding these tiny enzymes? It must be of a higher quasi intelligent species than DNA. No?


No. DNA contains the code for making all the proteins in your body, but is not the "be all and end all". There are sections of DNA that recruit machinery to transcribe it (which can be "blocked" to regulate transcription rate), and another code - the epigenome - which determines which genes can be transcribed/translated/made. ALL of this is in response to chemical messages in the body, which are produced in response to the environment, no "god" needed (or used). Life as we know it is just big ol' bags o' chemistry.
no fate
3 / 5 (2) May 27, 2014
ALL of this is in response to chemical messages in the body, which are produced in response to the environment, no "god" needed (or used). Life as we know it is just big ol' bags o' chemistry.


I will go you one further and cite that all reactions are charge dependant on the atomic level. Neurons work (fire) through ion transport across a membrane and charge differential.

"The global team tested the R-loop model using specially modified microscopes in which single DNA molecules are stretched in a magnetic field."

The only way this works is if the telomeres are actually charges on the ends of the molecule.
JVK
1 / 5 (5) May 27, 2014
Biophysical constraints on protein folding suggest that life is not just big ol' bags o' chemistry, and that anyone who suggests such a thing is not aware of any biologically plausible models in which ecological variation is linked to ecological adaptations.

People who cannot grasp the complexity of patterns that link physics, chemistry, and conserved molecular mechanisms to the molecular biology of life seem most likely to think in terms that dismiss anything currently known about how the epigenetic landscape is linked to the physical landscape of DNA in the organized genomes of species from microbes to man.

The idea that an asteroid somehow created chiral amino acids but left glycine to be substituted and stabilize the genomes of microbes and to also stabilize the decapeptide, GnRH in mammals, is one that I think serious scientists can most appreciate as the pseudoscientific nonsense of theorists who think in terms of randomness and evolution.
supamark23
5 / 5 (3) May 27, 2014
Biophysical constraints on protein folding suggest that life is not just big ol' bags o' chemistry, and that anyone who suggests such a thing is not aware of any biologically plausible models in which ecological variation is linked to ecological adaptations.

People who cannot grasp the complexity of patterns that link physics, chemistry, and conserved molecular mechanisms to the molecular biology of life seem most likely to think in terms that dismiss anything currently known about how the epigenetic landscape is linked to the physical landscape of DNA in the organized genomes of species from microbes to man.

The idea that an asteroid somehow created chiral amino acids but left glycine to be substituted and stabilize the genomes of microbes and to also stabilize the decapeptide, GnRH in mammals, is one that I think serious scientists can most appreciate as the pseudoscientific nonsense of theorists who think in terms of randomness and evolution.


bahahahahahahahahahahahahaha
supamark23
not rated yet May 27, 2014
ALL of this is in response to chemical messages in the body, which are produced in response to the environment, no "god" needed (or used). Life as we know it is just big ol' bags o' chemistry.


I will go you one further and cite that all reactions are charge dependant on the atomic level. Neurons work (fire) through ion transport across a membrane and charge differential.

"The global team tested the R-loop model using specially modified microscopes in which single DNA molecules are stretched in a magnetic field."

The only way this works is if the telomeres are actually charges on the ends of the molecule.


You know that DNA has an overall negative charge from all those phosphodiester bonds, right?
anonymous_9001
5 / 5 (1) May 27, 2014
Biophysical constraints on protein folding suggest that life is not just big ol' bags o' chemistry


This doesn't make any sense. None at all. Life is constrained by the properties of the chemicals that make it up, therefore life is not merely chemical-based? Is that supposed to mean something?

People who cannot grasp the complexity of patterns that link physics, chemistry, and conserved molecular mechanisms to the molecular biology of life seem most likely to think in terms that dismiss anything currently known about how the epigenetic landscape is linked to the physical landscape of DNA in the organized genomes of species from microbes to man.


People deny epigenetic control of transcription? Not as far as I know.

anonymous_9001
5 / 5 (2) May 27, 2014
The idea that an asteroid somehow created chiral amino acids but left glycine to be substituted and stabilize the genomes of microbes and to also stabilize the decapeptide, GnRH in mammals, is one that I think serious scientists can most appreciate as the pseudoscientific nonsense of theorists who think in terms of randomness and evolution.


"There's one achiral amino acid, therefore mutation and natural selection is unfounded."

Does not make sense. That conclusion does not follow from the premise.
JVK
1 / 5 (3) May 27, 2014
Ecological variation is linked to ecological adaptations via biophysically constrained nutrient-dependent protein folding that is manifested in the morphological and behavioral phenotypes of species from microbes to man via conserved molecular mechanisms of pheromone-controlled reproduction. The achiral amino acid that appears to stabilize the genome of 99% of extant or extinct bacteria and 100% of extant vertebrates is glycine, despite all of the obvious biodiversity.

Please tell us how mutations and natural selection contribute to biodiversity in species from microbes to man, or tell me what you think is wrong with my model of cause and effect.

Nutrient-dependent/pheromone-controlled adaptive evolution: a model.
http://www.ncbi.n...24693353

JVK
1 / 5 (3) May 27, 2014
Nutrient-dependent amino acid substitutions differentiate cell types and they are fixed in the organized genome of individuals with different cell types in different species. Fixation occurs via the physiology of reproduction, which is nutrient-dependent and pheromone-controlled.

Mutations are not fixed in the genome of C. elegans. Ivo Chelo explains: "Our data suggests that the value a new allele brings to the individuals is not fixed." http://www.scienc...5804.htm

"The patterns of synaptic connections perfectly mirror the fundamental differences in the feeding behaviours of P. pacificus and C. elegans", Ralf Sommer concludes. http://phys.org/n...ing.html

These reports link biophysically constrained nutrient-dependent pheromone-controlled biodiversity via epigenetic effects of olfactory/pheromonal input in species from microbes to man. I detailed how biodiversity arises in my published works.
johnhew
not rated yet May 28, 2014
I thought all the hoopla with glycine vs chiral amino acids was theorized to spring from the polarization of radiation?
JVK
1 / 5 (2) May 28, 2014
hoopla with glycine vs chiral amino acids


That's why I hoped that Luca Turin might comment on 5hmcs in our tweets, which would have moved us towards a collaborative effort. You and he have approached integration of physics and chemistry, but a more cohesive group will be needed to address the links to conserved molecular mechanisms accross species.

For example, I'm not familiar enough with physics to bring in the links from frugivory in bats to cell type differentiation protected from ionizing radiation by vitamin C and repair of damage that otherwise appears to result in genomic instability. However, Luca could probably link it via vibrations theory to the de novo creation of olfactory receptor genes (via Crawford's work on DHA and the quantum physics of cell membranes). Meanwhile, as I indicated, we have evolutionary theorists who think they are capable of intelligent discussion of biological facts --and they prevent intelligent discussion of my works.
JVK
1 / 5 (3) May 28, 2014
"About 400 millions years ago, a single substitution of the chiral amino acid in position 6 of GnRH in jawless fish by the achiral glycine facilitated formation of a type II' β-turn conformation of GnRH to allow close spatial interaction of these two functional elements."

http://www.ifzz.p...05〈=en

"...the surprising total conservation of GnRH II's primary structure, from bony fish to man, appears to be a result of the excellent coordinated evolutionary selection of amino acids participating in binding, activation and configuration such that its structure cannot be improved by substitution with any natural amino acid at any position."

If you're willing to use the 400 million years estimate of vertebrate genome stability, mutations and natural selection appear to make sense. If not, only nutrient-dependent pheromone-controlled ecological adaptations via amino acid substitutions from viruses to elephants makes sense.
JVK
1 / 5 (3) May 28, 2014
"The essential role of Gly-cisPro motif in chiral discrimination is also strongly indicated by its absolute invariance in all DTD sequences from eubacteria to higher eukaryotes (Figure 8C)."

http://elife.elif...2/e01519

See also: "Chirality Check" http://www.scienc...9.2.full

For a reality check see: http://sandwalk.b...ife.html

The link between asteroids, chirality, and life on earth seems as far-fetched as anything associated with mutations and natural selection in the context of biodiversity attributed to evolution. But some people really, really, really want to believe in pseudoscientific nonsense.

Many of them can be found on PZ Myers blog site. See http://freethough...t-737848
no fate
1 / 5 (1) May 28, 2014
"The global team tested the R-loop model using specially modified microscopes in which single DNA molecules are stretched in a magnetic field."

The only way this works is if the telomeres are actually charges on the ends of the molecule.


You know that DNA has an overall negative charge from all those phosphodiester bonds, right?

I do now. Thanks. You do realize that in order to magnetically "stretch" a molecule the field has to attract one pole and oppose the other,right?