Major breakthrough in precise genome editing with CRISPR

CRISPR-mediated genome editing has a major flaw: the nuclease cleaves the DNA at a specific site, but then the double strand break (DSB) is healed by an error-prone repair most of the time. This means, although site-specific, the genome editing is usually not precise, i.e. leads to random mutations at the site (owing to the DNA repair mechanism, non-homologous end-joining (NHEJ). However there is an existing possibility to carry out precise modifications, this is done by another pathway homology directed repair (HDR). During HDR, one utilizes an external DNA fragment, which has homology arms to the region facilitating to copy this region into the genome. However, HDR and NHEJ are competing with each other, NHEJ being the winner. Moreover, NHEJ is active in all cells at all time, but HDR is thought to be only effective in dividing cells in the S and G2 phase.

The group of Hidde Ploegh at Whitehead Institute at MIT, Boston, took a very straightforward approach by inhibiting NHEJ to favor HDR in cells expressing CRISPR/Cas9. This seems simple, but it's not, since most NHEJ inhibiting strategies makes cells really sick. By using an anti-cancer agent, Scr7, a commercially available NHEJ inhibitor, the authors were able to boost up HDR by 19-fold.

First, they analyzed CRISPR-expressing cell lines treated with Scr7. They introduced a site-specific STOP codon into the TSG101 gene and analyzed the presence of the STOP codon by simple PCR. Maximal HDR rate was detected to be about 3.1% for A549 cells and 19.1% for MelJuSo cells. Furthermore they were able to demonstrate successful insertion of a 800bp fragment (Venus) into a bone-marrow derived dendritic cell line, with efficacy up to 58%.

Next, they have looked at genome editing with HDR in mouse embryos, injected with Cas9, gRNA and HDR template. Surprisingly, mouse embryos treated with Scr7 not only showed higher frequency of HDR, but they showed the complete absence of NHEJ in 4 separately analyzed genes.

With permission from NPG, Maruyama T et al, Nat Biotechnol 2015

The current study provides a major step forward in increasing HDR over NHEJ in cell lines and particularly in mouse embryos, easing the development of precisely modified genetic mice. However, question remains, whether NHEJ could be inhibited and HDR could be carried out in non-dividing cells. I anticipate that increasing HDR frequency, compatible with potential human clinical applications, is going to be one of the major direction in the genome editing field.

Check out the paper here: http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3190.html

Combat HIV via the CRISPR system

With permission from NPG

It look like a real "cure" is underway for AIDS, a disease used to be one of the most fearsome one of the modern era. Just recently, Sangamo Biosciences received FDA approval to carry out clinical trials with zinc-finger nucleases to knock out the virus receptor on normal human hemopoietic stem cells. Now, it seems that the bacterial defense system, the CRISPR, could also be utilized to combat viral infection in human cells. In a recent paper, a group from La Jolla, CA developed a CRISPR strategy to attack invading HIV, making the antiviral tool expressing cells resistant to infection.

The authors first transduced 293T cells with replication incompetent lentiviruses carrying a GFP as a target for CRISPR. Not surprisingly disruption of GFP was readily detectable in Cas9/gRNA expressing cells. Novelty here is that it seems Cas9 can target the pre-integration viral genome, i.e. the human chromosomal environment is not required for action (as assayed by non-integrating lentiviruses). This is quite plausible, since the original bacterial CRISPR system indeed inactivates viral and plasmid DNA sequences, which have not been integrated into the host. Interestingly, CRISPR was able to be active in a high copy number system, projecting its usefulness in transgene animal models, where multiple copies of the transgene is present.

Also not surprisingly CRISPR was able to target HIV viral sequences in 293T.CD4.CCR5 cells, latently infected human T cell lines and differentiated human pluripotent stem cells. However, primary human T-cells were conferred only partially resistant to HIV infection.

This study is in correlation with other studies demonstrating antiviral effect of CRISPR against hepatitis B virus, papillomavirus, herpes simplex virus. I'm not sure how these findings are translatable to human clinical trials.

Full text of the paper here: Liao HK, Gu Y, Diaz A, Marlett J, Takahashi Y, Li M, Suzuki K, Xu R, Hishida T, Chang CJ, Esteban CR, Young J, Izpisua Belmonte JC. Use of the CRISPR/Cas9
system as an intracellular defense against HIV-1 infection in human cells. Nat Commun. 2015 Mar 10;6:6413. doi: 10.1038/ncomms7413.

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.

Genome editing meeting agenda for 2015

Here you can find information on conferences, symposia, workshops on CRISPR and genome editing for the rest of this year:

March 18-19, 2015: Genome editing applications, Boston, MA
IBC's Genome Editing Applications conference will showcase the most up-to-date therapeutic and biomedical applications emerging using CRISPRs, ZFNs, TALENs, AAVs and other genome engineering technologies. From validation of targeted cell lines and development of transgenic animal models to therapeutic uses in cell therapy, gene therapy and immunotherapy, this event will also highlight applications in drug target identification/validation and lead discovery and explore how these technologies are being used for whole-genome and functional screening as well as basic research applications.
http://www.ibclifesciences.com/GenomeEditing/overview.xml

March 23-24, 2015: CRISPR 2015 Oxford, UK, registration deadline March 22
Technology and Application Symposium - 23rd March, Sigma-Aldrich Technology Workshop - 24th March
http://lpmhealthcare.com/crispr-2015/

May 13-16, 2015: American Society of Gene & Cell Therapy (ASGCT) 18th Annual Meeting
Definitely will feature plenty of genome editing talks.
http://www.asgct.org/meetings-educational-programs/asgct-annual-meetings/2015-annual-meeting

June 18-20, 2015: CRISPR Conference 2015 New York, NY 
http://www.crispr2015.com/

July 20-24, 2015: IGI CRISPR Workshop: Routes to Designer Biology
The Innovative Genomics Initiative (IGI) is offering a 1-week lecture-based genome editing workshop at UC Berkeley on July 20-24, 2015. Scheduled presenters include Jennifer Doudna, Dana Carroll, Jonathan Weissman, Jacob Corn and others who will address specialized topics in genome editing and CRISPR/Cas9 research, including background and basic engineering, structures and mechanisms, multiplexing, bioinformatics, technology applications and bioethics. Small group sessions will enable direct interactions among students, lecturers and IGI staff to facilitate focused discussions on cutting-edge research experimental techniques, theory and applications. This workshop is designed for scientists in academia and industry who want to initiate or expand the use of CRISPR technology in their research. A working knowledge of basic molecular biology will be assumed. Applications from graduate students, postdoctoral fellows and more senior researchers are welcome.
http://innovativegenomics.org/crispr-workshop/

September 24 - 27, 2015: Genome Engineering: The CRISPR/Cas Revolution, Cold Spring Harbor Laboratory, NY, abstract deadline: Abstract Deadline: July 3, 2015
"The specific goal for this meeting is to foster fruitful and creative interactions between researchers interested in applying these systems to genome engineering and related advances in a wide variety of organisms, together with scientists studing the basic biology of CRISPR/Cas and related bacterial defense systems. The meeting will consist of six oral sessions and oneposter session/"
http://meetings.cshl.edu/meetings/2015/crispr15.shtml

If you find a meeting that is not included here, please let me know.

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.

Want to design a CRISPR for genome editing? Here are the PAM sites you need to keep in mind:

The PAM site is the only sequence requirement when designing a specific guide RNA for the CRISPR, i.e. only sites in the genomic DNA next to a specific PAM motif could be targeted and edited. The PAM site is in the genomic DNA, not in the guide RNA. The presence or absence of this motif next to your favorite sequence is one of the major limitations of the CRISPR/Cas9 system. If a Cas9 would be utilized to cleave an allele with a mutation, the mutation must be next (within preferably 10nt) to a PAM site.

(The PAM site is needed for the bacterial CRISPR system to discriminate the bacterial and viral genome, since the PAM site is absent from the former but present in the latter.)

Here are the PAM sites for the currently used Cas9 enzymes, check if you find a good one next to your sequence of interest:

R: A or G, W: A or T
*Be careful with non-canonical PAM sites, at some sites there might be no activity
**Not yet available, will be available soon

1. 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. doi: 10.1126/science.1231143.
2. Zhang Y, Ge X, Yang F, Zhang L, Zheng J, Tan X, Jin ZB, Qu J, Gu F. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. Sci Rep. 2014 Jun 23;4:5405. doi: 10.1038/srep05405.
3. Friedland AE, Sousa A, Collins M. et al. S. aureus Cas9: and alternative Cas9 for genome editing applications, Editas Medicine, http://paperzz.com/doc/3029869/read-more---editas-medicine
4. Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15644-9. doi: 10.1073/pnas.1313587110.
5. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 2011 Nov;39(21):9275-82. doi: 10.1093/nar/gkr606.

Advances in Genome Engineering Using CRISPR-Cas9 workshop @ Broad, March 11th, 2015

The Broad Institute organizes a workshop in recent developments in the CRISPR system.
More info and registration: https://www.broadinstitute.org/partnerships/education/broade/broad-workshops#

Rare hemoglobin disorders remain major target diseases to be corrected with genome editing systems

Recently, Sangamo Biosciences received green light from FDA to start clinical trials on beta-thalassemia. In their approach, finc-finger nucleases shall disrupt transcriptional regulators to switch the production from mutated adult hemoglobin back to normal fetal hemoglobin. Now, another company, Editas Medicine reported successful in vivo gene repair for sickle cell anemia at the Keystone Symposium for Genomic Instability and DNA Repair in Whistler, British Columbia, Canada. This hereditary hemoglobinopathy is characterized by a mutation in the hemoglobin beta (HBB) gene leading to abnormal red blood cell morphology under certain circumstances. The presented mechanism for gene repair is thought to be gene conversion, i.e. self-repair of the CRISPR targeted allele utilizing a different, but closely related gene as a repair template. The efficiency and precise mechanism remains a question. “These data suggest gene conversion as a possible new approach to genomic repair for certain kinds of genetic mutations,” said Katrine Bosley, chief executive officer, Editas Medicine. “While the results are early and further work is needed to see if this approach could be used therapeutically, the data exemplify Editas’ commitment to explore and develop the full potential of genome editing to treat a broad range of genetically driven diseases.”
Source: http://www.editasmedicine.com/documents/Editas%20Keystone%20Data%20PR%20030115%20FINAL%20.pdf