CRISPR happenings - Cold Spring Harbor Laboratory hosts the 'Genome Engineering: The CRISPR/Cas9 revolution' meeting between September 24-27, 2015. The meetings is organized by Jennifer Doudna, (University of California, Berkeley/HHMI), Maria Jasin (Memorial Sloan Kettering Cancer Center, NY) and Jonathan Weissman (UCSF/HHMI).

Oral sessions will feature CRISPR Biology, DNA Repair/Genome Editing, Human Genome Engineering, Model Organisms/Plants
Technology Development, Stem Cells/Cancer.

Abstract submission deadline: July 3, 2015. Stipends might be available for students.

More info here: http://meetings.cshl.edu/meetings.aspx?meet=crispr&year=15

2015 American Society of Gene and Cell Therapy Annual meeting in New Orleans, LA - Highlights - Long-term engraftment of zinc finger modified T-cells in HIV infection

Sangamo Biosciences (Richmond, CA, USA) presented novel clinical data regarding the SB-728-T-cell program for viral load control in chronic HIV infection. In this open-label phase 1 clinical trial, nine patients with low CD4+ T cell counts (200-500 cells/mm3) received 10-30 billion autologous, zinc finger nuclease modified CCR5 knockout T-cells.
Interestingly, modified T-cells were still detectable in the circulation after 3 years and generated long-lived T-memory stem cells. The authors demonstrated a sustained increase of CD4+ T cell counts in all treated subjects. T-memory stem cell count correlated with the decay of viral load.

Taken together, the SB-728-T-cell infusion unexpectedly resulted in very long term T-cell reconstitution, owing to the transformation of the zinc-finger edited T-cells to long lived memory stem cells. The program is now in Phase II, while a similar hemopoietic stem cell approach is already in phase I.

CRISPR corrects gene defect in Fanconi-anemia

A new paper came out on using CRISPR to edit and correct the FANCC gene, responsible for Fanconi anemia, a rare genetic disease affecting DNA repair. A point mutation leads to a cryptic splice site and in frame deletion of an exon in the FANCC gene. These patients exhibit hematological malignancies, solid tumors and often bone marrow failure. Currently patients are treated with allogenic hematopoietic cell transplantation. Obviously, ex vivo gene correction of the mutated gene is a feasible option in the context of autologous hematopoietic transplantation.

Key facts:
1. Cas9 nickase is less effective than WT Cas9
2. Cas9 nickase leads more likely to homology directed repair (HDR) events than the WT Cas9.
3. No off-target effects were found in the genome
4. Function of FANCC gene was restored in patient derived fibroblasts.

The group of Jakub Tolar (University of Minnesota) designed guide RNA-s against the FANCC gene and analyzed NHEJ (random mutations as a consequence of successful CRISPR action) on 293T cells and patient-derived fibroblasts. Using the not very sensitive SURVEYOR assay, random mutations were obvious in 293T cells, but hardly detectable in the fibroblasts, most likely because of inefficient transfection and low sensitivity of the SURVEYOR mutation detection assay.

Next, they took advantage of the traffic light reporter system to assess HDR (homology directed repair, "real" correction) and NHEJ rates in 293T cells. The traffic light system is a very creative way to detect gene specific CRISPR action: the sequence of interest is inserted upstream of an out of frame mCherry, which is put back in frame after NEHJ (red cells); while HDR oligo mediated recombination will generate a functional GFP (green cells). Using this approach, the authors showed that the WT Cas9 nuclease is much more active than the nickase (assessed by overall higher NEHJ and HDR), but, as reported previously, the nickase lead to preferential HDR over NEHJ.

Off-target effects were measured by either the SURVEYOR assay on predicted sites, but also using an integrase defective lentiviral (IDLV) capture assay, developed by a group in Heidelberg. The principle is that IDLV can only integrate at double strand breaks, so if the CRISPR has off target effects, the lentivirus could be amplified from the site using primers specific for the viral DNA. Using the IDLV method, no off-target effects were found for Cas9 and Cas9 nickase. We have to keep in mind however that the IDLV method is a less sensitive, somewhat sequence biased approach compared to the recently published method by K. Joung (GUIDE-Seq).

Finally, FANCC gene was corrected using HDR repair template containing puromycin selectable marker. The nickase lead higher number of corrected clones than the WT Cas9. In the corrected clones, the mRNA contained the skipped 4. exon, and the protein functionality was restored in a histone phosphorylation assay. It is worth to note that it is pretty challenging to carry out genome editing in FANCC cells, since the mutated protein is involved in DNA repair (and possibly HDR). Therefore the successful strategy was to use a plasmid homology template, which encoded for the FANCC cDNA.

In summary, this paper provides evidence that Fanconi anemia could be a target for genome editing. The next step is to carry out the precise gene correction in hemopoietic stem cells, assess in vivo rescue of function and to characterize off-target effects in more detail.

Link to the paper: Osborn MJ, Gabriel R, Webber BR, DeFeo AP, McElroy AN, Jarjour J, Starker CG, Wagner JE, Joung JK, Voytas DF, von Kalle C, Schmidt M, Blazar BR, Tolar J. Fanconi Anemia Gene Editing by the CRISPR/Cas9 System. Hum Gene Ther. 2015 Feb;26(2):114-26.

Speeding up human evolution via genome editing?

I came across this scary article recently. By reading the article you can get a sense how astonishingly close we are to create genetically modified human embryos. Fact is, the first gene modified monkeys were born in China last year, and rumors suggest the first modified human embryos are already created somewhere in China too. Implanting these embryos would give rise to the first human beings, whose code of life has been directly manipulated. And there is no doubt that using CRISPR technology this seems simple. I think - being a researcher - I could learn it in a few months or so. It's pretty much straightforward. But the question remains, why would anyone on earth do this?

 It is important to emphasize that medical reasons to gene edit embryos are practically non-existing. This must be done on in vitro fertilized embryos, which could be screened for genetic diseases anyway (this is a already a 'treatment option' for those couples who have certain mutant genes). In this case, only the normal gene carrying embryos would be implanted into the mother, avoiding the need for genome editing.  

So gene editing would only help those couples who altogether carry at least 3 gene defects on the same gene! (except for the very unlikely case of carrying 2 dominantly inherited alleles in the same parent). This is a very rare case, so companies try to raise money for another potential application - to boost up intelligence or strength. In a recent survey, 15% of Americans would agree to carry out gene modifications on embryos in order to make the baby more intelligent. I'm not expecting that such a treatment option would be available in the near future, given the complexity of intelligence development. But in the distant future, targeted gene modifications will be feasible in favor of accelerated human evolution.

Check out the article here.

Colon carcinoma development in the dish 

With permission from NPG, licence number:  3575540372115

Researchers from Japan were able to model the development of colon cancer in intestinal organoids. CRISPR was used to disrupt APC, SMAD4 and TP53 tumor suppressor genes and to introduce tumor specific mutations into KRAS and PIK3CA in intestinal organoids isolated from human patients. Most interestingly engineered normal tissue was not converted to highly invasive tumor. On the contrary, when the engineering was done in chromosome instabile adenomas, the organoids formed macrometastatic colonies in mice. This study shows the versatility of genome editing to model a highly complex disease process.

Matano M, Date S, Shimokawa M, Takano A, Fujii M, Ohta Y, Watanabe T, Kanai T,
Sato T. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of
human intestinal organoids. Nat Med. 2015 Feb 23.

http://www.nature.com/nm/journal/vaop/ncurrent/full/nm.3802.html

Genome editing and the CRISPR

Genome editing: break and repair

In the modern era of genetics, it is simple to read the human genome, determine every single nucleotide in your cells can be done in a few days at an relatively low cost. However it is extremely challenging to change something in the DNA, e.g. to correct a disease causing mutation. This molecule is extremely stable and well-protected from any interventions, so until recently it was hardly possible to ’edit’ the genome. Editing means everything that writers or publishers do with a written text: cutting, pasting  or changing letters.  Genome editing is similarly done with the four  nucleotides (A, G, C, T) in the DNA. Genome editing is not a completely new technolgy researchers have been using other enyzmes (like the so-called zinc  finger nucleases and the TALENs) to perform genome editing. But there is a huge differnce between the these and the novel technology, CRISPR (the abbreviation is very complicated: Clustered Regularly Interspaced Short Palindromic Repeats) The former technologies use peptide decoders to find the certain part in the long DNA that has to be modified and therefore it is challenging to figure out what peptide is the one that exclusively binds to my favourite sequence. But CRISPR is a real gift from the nature: it uses a complimentary RNA, which will just pair with one strand of the DNA. As you may know, the whole genome has been already deciphered, we know the sequence of the human genome. If it was written on sheets, the tower of books would be 169 meter high!

So, if the genome sequence is known, then it is simple to design the RNA (this will be called the guide RNA, as this guides the editing enzyme to the genomic region) that is complimentary to the DNA. As you know: adenin [A] pairs with timine [T], guanine [G] with cytosine [C] and vice versa. Let’s see: here is a diseases gene sequence: AGCTGTGCTGTCGATGC. I want to edit this. So first I have to direct the editing enzyme (this is called the Cas9) to this sequence with the guide RNA, which has the sequence of: TCGACACGACAGCTACG. So look at this!

The gene to be edited: AGCTGTGCTGTCGATGC

The guide:                       TCGACACGACAGCTACG

These are pairs! So to be simple: the guide RNA can select the region of interest from 3 billion base pairs quickly and effectively.

OK, so we have something that selects a gene of interest in the genome. The next question is: how this part of the genome will be edited? Basicly, the Cas9 protein will cleave the DNA and create a double strand break (so it cuts the DNA to two parts). Cells doesn’t like this, a break in the DNA is a nightmare for them, so they want to utilize one of many repair mechanisms to rejoin the injured DNA. Among these is the so-called NHEJ – non-homologus end joining. This repair is an error-prone repair, which will create mutations (some difference compared to the original), otherwise the Cas9 enzymewill cut it again and again. So it has to be some change in the sequence so that the RNA guide will not recognize it anymore. This NHEJ can kick out a gene and inactivate it. So let’s say there is a mutation in a gene that leads to an abnormal protein that has abnormal function, like a tumor formation. We have two copies of each gene (one from the mother and one from the father), but even if we have ONE normal copy, there is that bad gene that drives tumor growth. This is called the DOMINANT effect, with one bad copy, there is still disease. But using CRISPR this bad gene coud be knocked out, so the normal copy will take over and the disease can be cured! Forever since it the DNA is modified!

But you can say, where is the editing? Where is the real correction? Fortunatley this is also possible. If you happen to deliver a repair template to the cells (from which the DNA could be repaired) you can insert anything into the genome, exactly at the cut site, where the DNA is broken. This is called the homology directed repair (HDR). So if someone has a so-called RECESSIVE disorder (when both copies of the gene is mutated), the gene can be corrected with this method.

And applications are indefinite: it is feasible to do it in plants, bacteria, or other animals to cure diseases, or to create even new lifestyles. This system can be also used as a novel technology to study the function of genes. I will tell you even more, if you come back to this site!