Friday, November 08, 2013

CRISPR precision Genetic Engineering

CRISPR precision Genetic engineering .

Andre Willers
8 Nov 2013
“I expected this , but not so soon .”  Epitaph
Synopsis :
Precision additions and elisions can be done on any species’ genome , including vectors .

Discussion :
1.A fundamental breakthrough . Gene therapy even on mature organisms is now possible .
2.Immortality , youth , regeneration et blah , blah , blah .
3.One teensy , weensy problem , though . The epigenetic system , as well as bacterial and viral interactions .
4.We can do it , but like the Sorceror’s Apprentice , do not know enough .
5.But simple genetic diseases can be cured by correcting the genome .
6.Initially only in fertilized cells .
7.But vectors like viruses or tailored bacterio-viri can be designed and built to change the genome in a mature organism .
8.It is very easy to use . High-school level .
9.Expect a reprise of the copy-paste kiddies in computer viruses . Only now they will be biological , or hybrids .
10.The Whitehats will contain the Blackhats , or everybody will die . The contest will never end .
11.An interesting solution to the overpopulation and resources problem : Everybody ends up CRISPR .
12. The Technology itself :
See Appendix A
13. A bit more detail
See Appendix B
14.Synergism :
Computers , bio-3D printers , CRISPR biochips , Genetic  algorithms  , half-baked humans and we are off to the races .
15. Singularity effects :
By 2100 AD you will have to look long and hard to find a Homo Sapiens .
Even stay-behinds will have to extensively modified to survive what is coming .
Cheer up . Everybody used to die .

Appendix A
Exclusive: 'Jaw-dropping' breakthrough hailed as landmark in fight against hereditary diseases as Crispr technique heralds genetic revolution
Development to revolutionise study and treatment of a range of diseases from cancer, incurable viruses such as HIV to inherited genetic disorders such as sickle-cell anaemia and Huntington’s disease


Thursday 07 November 2013
A breakthrough in genetics – described as “jaw-dropping” by one Nobel scientist – has created intense excitement among DNA experts around the world who believe the discovery will transform their ability to edit the genomes of all living organisms, including humans.
Click image above to enlarge graphic
The development has been hailed as a milestone in medical science because it promises to revolutionise the study and treatment of a range of diseases, from cancer and incurable viruses to inherited genetic disorders such as sickle-cell anaemia and Down syndrome.
For the first time, scientists are able to engineer any part of the human genome with extreme precision using a revolutionary new technique called Crispr, which has been likened to editing the individual letters on any chosen page of an encyclopedia without creating spelling mistakes. The landmark development means it is now possible to make the most accurate and detailed alterations to any specific position on the DNA of the 23 pairs of human chromosomes without introducing unintended mutations or flaws, scientists said.
The technique is so accurate that scientists believe it will soon be used in gene-therapy trials on humans to treat incurable viruses such as HIV or currently untreatable genetic disorders such as Huntington’s disease. It might also be used controversially to correct gene defects in human IVF embryos, scientists said.
Until now, gene therapy has had largely to rely on highly inaccurate methods of editing the genome, often involving modified viruses that insert DNA at random into the genome – considered too risky for many patients.
The new method, however, transforms genetic engineering because it is simple and easy to edit any desired part of the DNA molecule, right down to the individual chemical building-blocks or nucleotides that make up the genetic alphabet, researchers said.
“Crispr is absolutely huge. It’s incredibly powerful and it has many applications, from agriculture to potential gene therapy in humans,” said Craig Mello of the University of Massachusetts Medical School, who shared the 2006 Nobel Prize for medicine for a previous genetic discovery called RNA interference.
“This is really a triumph of basic science and in many ways it’s better than RNA interference. It’s a tremendous breakthrough with huge implications for molecular genetics. It’s a real game-changer,” Professor Mello toldThe Independent.
“It’s one of those things that you have to see to believe. I read the scientific papers like everyone else but when I saw it working in my own lab, my jaw dropped. A total novice in my lab got it to work,” Professor Mello said.
In addition to engineering the genes of plants and animals, which could accelerate the development of GM crops and livestock, the Crispr technique dramatically “lowers the threshold” for carrying out “germline” gene therapy on human IVF embryos, Professor Mello added.
The new method of gene therapy makes it simple and easy to edit any desired part of the DNA molecule (Getty Creative)
Germline gene therapy on sperm, eggs or embryos to eliminate inherited diseases alters the DNA of all subsequent generations, but fears over its safety, and the prospect of so-called “designer babies”, has led to it being made illegal in Britain and many other countries.
The new gene-editing technique could address many of the safety concerns because it is so accurate. Some scientists now believe it is only a matter of time before IVF doctors suggest that it could be used to eliminate genetic diseases from affected families by changing an embryo’s DNA before implanting it into the womb.
“If this new technique succeeds in allowing perfectly targeted correction of abnormal genes, eliminating safety concerns, then the exciting prospect is that treatments could be developed and applied to the germline, ridding families and all their descendants of devastating inherited disorders,” said Dagan Wells, an IVF scientist at Oxford University.
“It would be difficult to argue against using it if it can be shown to be as safe, reliable and effective as it appears to be. Who would condemn a child to terrible suffering and perhaps an early death when a therapy exists, capable of repairing the problem?” Dr Wells said.
The Crispr process was first identified as a natural immune defence used by bacteria against invading viruses. Last year, however, scientists led by Jennifer Doudna at the University of California, Berkeley, published a seminal study showing that Crispr can be used to target any region of a genome with extreme precision with the aid of a DNA-cutting enzyme called CAS9.
Since then, several teams of scientists showed that the Crispr-CAS9 system used by Professor Doudna could be adapted to work on a range of life forms, from plants and nematode worms to fruit flies and laboratory mice.
Earlier this year, several teams of scientists demonstrated that it can also be used accurately to engineer the DNA of mouse embryos and even human stem cells grown in culture. Geneticists were astounded by how easy, accurate and effective it is at altering the genetic code of any life form – and they immediately realised the therapeutic potential for medicine.
“The efficiency and ease of use is completely unprecedented. I’m jumping out of my skin with excitement,” said George Church, a geneticist at Harvard University who led one of the teams that used Crispr to edit the human genome for the first time.
“The new technology should permit alterations of serious genetic disorders. This could be done, in principle, at any stage of development from sperm and egg cells and IVF embryos up to the irreversible stages of the disease,” Professor Church said.
David Adams, a DNA scientist at the Wellcome Trust Sanger Institute in Cambridge, said that the technique has the potential to transform the way scientists are able to manipulate the genes of all living organisms, especially patients with inherited diseases, cancer or lifelong HIV infection.
“This is the first time we’ve been able to edit the genome efficiently and precisely and at a scale that means individual patient mutations can be corrected,” Dr Adams said.
“There have been other technologies for editing the genome but they all leave a ‘scar’ behind or foreign DNA in the genome. This leaves no scars behind and you can change the individual nucleotides of DNA – the ‘letters’ of the genetic textbook – without any other unwanted changes,” he said.
Timeline: Landmarks in DNA science
Restriction enzymes: allowed scientists to cut the DNA molecule at or near a recognised genetic sequence. The enzymes work well in microbes but are more difficult to target in the more complex genomes of plants and animals. Their discovery in the 1970s opened the way for the age of genetic engineering, from GM crops to GM animals, and led to the 1978 Nobel Prize for medicine.
Polymerase chain reaction (PCR): a technique developed in 1983 by Kary Mullis (below, credit: Getty) in California allowed scientists to amplify the smallest amounts of DNA – down to a single molecule – to virtually unlimited quantities. It quickly became a standard technique, especially in forensic science, where it is used routinely in analysing the smallest tissue samples left at crime scenes. Many historical crimes have since been solved with the help of the PCR test. Mullis won the Nobel Prize for chemistry in 1993.

RNA interference: scientists working on the changing colour of petunia plants first noticed this phenomenon, which was later shown to involve RNA, a molecular cousin to DNA. In 1998, Craig Mello and Andrew Fire in the US demonstrated the phenomenon on nematode worms, showing that small strands of RNA could be used to turn down the activity of genes, rather like a dimmer switch. They shared the 2006 Nobel Prize for physiology or medicine for the discovery.
Zinc fingers: complex proteins called zinc fingers, first used on mice in 1994, can cut DNA at selected sites in the genome, with the help of enzymes. Another DNA-cutting technique called Talens can do something similar. But both are cumbersome to use and difficult to operate in practice – unlike the Crispr technique.
Click HERE to see a video of how the Crispr system derived from bacteria works on human cells to correct genetic defects
Appendix B
Addgene's Guide to CRISPR Technologies

Addgene is a global, non-profit plasmid repository dedicated to making it easier for scientists to share.
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CRISPR Plasmids


Zinc Finger Plasmids

Overview of CRISPR Technology

Figure 1: The current genome engineering technologies allow scientists to introduce double stranded breaks at specific sequences. Learn more on Addgene's Genome Engineering page.

The last few months have been an exciting time for genome engineering technology. A new system offers the first alternative to the current protein-based targeting (TALEN and Zinc Finger) methods used to specify a gene (or other DNA sequence). This new system uses a short RNA to guide a nuclease to the DNA target. This is CRISPR technology (Figure 1).

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) system was first discovered in bacteria and functions as a defense against foreign DNA, either viral or plasmid. So far three distinct bacterial CRISPR systems have been identified, termed type I, II and III. The Type II system is the basis for the current genome engineering technology available and is often simply referred to as CRISPR.

In bacteria, the endogenous CRISPR/Cas system targets foreign DNA with a short, complementary single-stranded RNA (CRISPR RNA or crRNA) that localizes the Cas9 nuclease to the target DNA sequence. The DNA target sequence can be on a plasmid or integrated into the bacterial genome. The DNA target also does not need to be unique and can appear in multiple locations, all of which will be targeted by the Cas9 nuclease for cleavage. The crRNA can bind on either strand of DNA and the Cas9 will cleave both strands (double strand break, DSB). The DSB results in the silencing of that DNA sequence.

The crRNA targeting sequences are transcribed from DNA sequences known as protospacers. Protospacers are clustered in the bacterial genome in a group called a CRISPR array. The protospacers are short sequences (~20bp) of known foreign DNA separated by a short palindromic repeat and kept like a record against future encounters. To create the CRISPR targeting RNA (crRNA), the array is transcribed and the RNA is processed to separate the individual recognition sequences between the repeats. In the Type II system, the processing of the CRISPR array transcript (pre-crRNA) into individual crRNAs is dependent on the presence of a trans-activating crRNA (tracrRNA) that has sequence complementary to the palindromic repeat. When the tracrRNA hybridizes to the short palindromic repeat, it triggers processing by the bacterial double-stranded RNA-specific ribonuclease, RNase III. Any crRNA and the tracrRNA can then both bind to the Cas9 nuclease, which then becomes activated and specific to the DNA sequence complimentary to the crRNA (Figure 2).

There are some restrictions to this new genome engineering system. Any potential target sequence must have a specific sequence on its 3’ end (the protospacer adjacent motif, PAM). The PAM is present in the DNA to be degraded but not the crRNA that’s produced to target it. The Type II CRISPR system is currently limited to target sequences that are N12-20NGG. Where NGG represents the PAM. Additionally, it is hypothesized that certain target sequences are believed to be problematic due to the RNA secondary structure they form. Interactions between the Cas9, tracrRNA and crRNA secondary structures are still poorly understood.

Figure 2: An overview of the endogenous Type II bacterial CRISPR/Cas system. Within the bacterial genome, a CRISPR array contains many unique protospacer sequences that have homology to various foreign DNA (e.g. viral genome). Protospacers are separated by a short palindromic repeat sequence. (A) The CRISPR array is transcribed to make the pre-CRISPR RNA (pre-crRNA). (B) The pre-crRNA is processed into individual crRNAs by a special trans-activating crRNA (tracrRNA) with homology to the short palindromic repeat. The tracrRNA helps recruit the RNAse III and Cas9 enzymes, which together separate the individual crRNAs. (C) The tracrRNA and Cas9 nuclease form a complex with each individual, unique crRNA. (D) Each crRNA:tracrRNA:Cas9 complex seeks out the DNA sequence complimentary to the crRNA. In the Type II CRISPR system a potential target sequence is only valid if it contains a special Protospacer Adjacent Motif (PAM) directly after where the crRNA would bind. (E) After the complex binds, the Cas9 separates the double stranded DNA target and cleaves both strands after the PAM. (F) The crRNA:tracrRNA:Cas9 complex unbinds after the double strand break.

CRISPR Systems Available at Addgene

The CRISPR systems that several research groups have developed for use in eukaryotic cells use a variation of a bacterial Cas9 nuclease that has been codon-optimized for their desired cell type. Additionally, a number of research groups have successfully demonstrated the effectiveness of a single fused crRNA-tracrRNA construct that functions with their codon-optimized Cas9 (Figure 3). This single RNA is often referred to as a guide RNA or gRNA. Within a gRNA, the crRNA portion is identified as the ‘target sequence’ and the tracrRNA is often referred to as the ‘scaffold’. This system has been shown to function in a variety of eukaryotic cells, including human, zebra fish and yeast. And for all of you interested in editing bacterial genomes, customized bacterial CRISPR systems have been created by the Doudna lab and the Marraffini lab to allow you to alter your favorite bacterial gene or DNA element.

There are many available online resources for helping scientists determine suitable target sites in their desired DNA sequence. The Church lab has published a bioinformatically generated list of ~190,000 potential gRNAs, targeting more than 40% of human exons. Scientists can search this list for their gene of interest. The Zhang lab has a similar online resource, which is home to a detailed background section on CRISPR technology, a discussion of the reagents in their system, and a tool for identifying potential targets in a variety of different species. The Joung lab also has a an online resource that includes a tool first developed for identifying potential Zinc Finger targets, then later modified to also identify TALEN array binding sites and is now also capable of identifying CRISPR target sites, all based on a user generated input sequence. There are other online, open access databases (such as designed to help scientists find CRISPR targeting sites in a wide range of species and generate the appropriate crRNA sequence.

Figure 3: A simplified overview of the various engineered CRISPR technologies created from the bacterial Type II system. (A) A codon-optimized version of the Cas9 nuclease with a Nuclear Localization Signal (NLS) is expressed from an appropriate promoter. The Cas9 has been optimized for expression in various eukaryotic cell types including human. (B) A customizable DNA element that allows for the transcription of crRNA-tracrRNA fused hybrid RNA, often referred to as a guide RNA (gRNA). Generally the sequence to be targeted (protospacer) is inserted into the gRNA expression plasmid by synthesizing complementary oligos with the appropriate restriction site overhangs on the 5’ and 3’ ends. (C) Both the Cas9 expression plasmid and gRNA expression plasmid are then transfected into the target cell. In some cases a single plasmid containing two expression cassettes can be used. The single gRNA binds with and activates the codon-optimized Cas9 nuclease.

While the specifics differ between the various engineered CRISPR systems, the overall methodology is similar; a scientist interested in using CRISPR technology to target a DNA sequence (identified using one of the many available online tools) needs only to insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid. The gRNA expression plasmid contains the PAM sequence, a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter and necessary elements for proper processing in eukaryotic cells. Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded DNA and then cloned into the gRNA expression plasmid. Double transfection of the gRNA expression plasmid and the Cas9 expression plasmid into your cells is all that is required.

Addgene is already the go-to source for the popular genome engineering kits utilizing TALEN technology and is now your source for the latest genomic engineering technology, CRISPRs. We strive to provide scientists all over the world with the opportunity and the means to share their work as easily as possible with the global community. Learn more about the CRISPR plasmids available at Addgene.

Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science 2013 Feb 15;339(6121):823-6.
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012 Aug 17;337(6096):816-21.
Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 2013 Mar;31(3):227-9.
Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 2013 Mar;31(3):233-9.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science 2013 Feb 15;339(6121):819-23


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