Crystal structure of Staphylococcus aureus Cas9 has been revealed

Feng Zhang (MIT, Cambridge) and a group from Tokyo provided 2.7 Å resolution structures for the smaller Cas9 variant from S. aureus. This Cas9 gained attention owing to it's size compatible with adeno-associated viral vectors. The PAM site (DNA recognition site) of this variant is 'TTGAAT' or 'TTGGGT', which is however more complex than the S. pyogenes 'NGG' PAM site. Interestingly, the S. aureus and the S. pyogenes Cas9 share
only 17% sequence identity, but have similar overall domain organization. Structural information enabled rational design of S. aureus Cas9-based transcriptional activators and split Cas9 design.





Link to paper: http://www.cell.com/cell/abstract/S0092-8674(15)01020-X

Zinc-finger nucleases facilitate in vivo integration of transgenes into the albumin locus

A group from the Children's Hospital of Philadelphia published a unique way to ameliorate hereditary bleeding disorders and enzyme deficiencies. Katherine A. High and her group utilized adeno-associated virus 8 (AAV8) to deliver a pair of zinc-finger nucleases (ZFNs) along with an interchangeable transgene to the liver. The transgene cassette is promoterless but is flanked by terminal regions homologous to the albumin gene. Upon ZFN cleavage, the transgene integrates with low efficiency, but under a very strong promoter, which leads to phenotype correction in a model of hemophilia A and hemophilia B. The correction of hemophilia A is particularly significant, since the gene encoding for factor VIII is larger than the AAV capacity. Thus, the most important advantage of this current method is that it increases AAV coding capacity by obviating the need of the promoter. In contrast to Mark Kay's recent study in Nature, this study did not observe any gene expression in the sole presence of an AAV encoding for the promoterless transgene. The authors noted some off-target effects, which might add up over time, given the long-term expression of ZFNs from an AAV episome.

Read the paper on the Blood journal website: http://www.bloodjournal.org/content/early/2015/08/20/blood-2014-12-615492.long?sso-checked=true

Parasites are targets for genome editing

There was a paper last week in Nature by the Striepen lab at the University of Georgia, showing that the well-known CRISPR system could be used to study an important human pathogen, Cryptosporodium parvum. The protozoan parasite causes diarrhea in children and in immunocompromised individuals (the image shows the parazites in the epithelial vacuoles of the intestine). Studying this parasite remains challenging due to lack of efficient propagation methods, simple animals models and molecular genetic manipulation techniques. Despite inefficient transfection, CRISPR/Cas9 along with a drug resistance cassette, rendered the parazites drug-resistant. Interestingly, non-homologous end-joining, a common error-prone DNA repair mechanism in higher organisms, is completely absent in Cryptosporodium. In contrast, homology-directed repair can lead to modifications in the genome when linked to Cas9 nuclease activity. The advance of this technique is that genetically modified parasites could be propagated and placed under selection pressure and in vivo. This would faciliate drug screening, drug target validation, but might also lead to the development of genetically modified, attenuated parasites for vaccination.

You can access the paper here: http://www.nature.com/nature/journal/v523/n7561/full/nature14651.html

New high-throughput analysis reveals parameters affecting guideRNA efficiency

A recent report in Nature Methods by one of the leading geneticist, George Church, unveils factors that affect genome editing efficiency with CRISPR. The group from the Wyss Institute and Harvard Medical School employed a library-on-library approach to figure out why certain gRNAs are more effective than others. They carried out two separate experiments; first they transfected cells with a target lentiviral library as well as a gRNA plasmid library, which enabled the scientists to analyze how a certain sequence affect genome editing rates. Not quite surprising, there was a massive variation among different gRNAs at different targets, but the most important base was found to be the PAM adjacent base, with G being the most efficient and T least preferred. Next, they transfected the gRNA plasmid library into 293T cells and looked at endogenous genome editing. This experiment allowed to analyze the effect of epigenetic parameters on genome editing efficiency. Indeed, there was a robust correlation between DN-ase I hypersensitive sites (i.e. more open chromatin, transcriptionally active regions) and editing efficiency. Finally, it is interesting to note that there was no tangible relationship between gRNA effectiveness and off-target activity, moreover some of the most active gRNAs had the least off-target effects.

The group made a prediction algorithm available
online: https://crispr.med.harvard.edu/sgRNAScorer/

Protospacer Workbench - an offline software for for rapid sgRNA design and off-target prediction

An offline tool available for MacOS X system is now available for free at http://www.protospacer.com/. The software combines the advantages of various CRISPR design algorithms. It features single or paired target design of gRNAs for which cleavage efficiency is estimated based on the Doenech score and off-target sites are predicted and validated. The software can handle very large genomes, custom sequences or user defined genes. gRNA design is only available for S. pyogenes Cas9 for N(20)NGG sites. It has a graphical user interface and does not require programming skills.  The workbench also connects to the IGV viewer.
Source: http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3291.html

Chemically modified guide RNAs facilitate genome editing in primary human cells 

A recent work from the Porteus lab (Stanford, California) presents the use of chemically altered gRNAs to improve low efficiency genome editing rates in primary human T cells and CD34+ hemopoietic stem cells. Modified sugar-phosphate backbone renders the gRNA more stable in cells leading to enhanced gene engineering, including more efficient non-homologous end joining and homology directed repair rates. These gRNAs have a particular advantage over unmodified gRNAs when the system is delivered as a ribonucleoprotein particle (RNP, i.e. Cas9 protein and gRNA) or as pure RNAs (Cas9 mRNA and gRNA), advantages over plasmid DNA expressed Cas9 are less clear. The authors claim there is somewhat better on/off target ratio with modified gRNAs, but we have to be cautious with this: the authors modified the terminal bases on the gRNA, perhaps functionally truncating them. Truncated gRNAs have significantly higher specificity according to the paper of from the K. Joung lab. Nevertheless, this system could be highly advantageous when working with primary cells, where genome editing rates are relatively low.

If you are interested in the paper, you can find it here.

Optical control of genome editing

The group of Moritoshi Sato (University of Tokyo) reported a novel, photoactivatable Cas9 variant (paCas9) for spatiotemporally regulated genome editing. Researchers made a split Cas9, with fragments fused to 'Magnet' dimerization domains. Upon blue light, Magnet domains bring together Cas9 components and reconstitute genome editing activity. The system is a little bit less effective than wtCas9, but it's activation is reversible and could be controlled in space. The authors provided evidence that the system works also with Cas9-nicakse and also with dead Cas9 (dCas9) for reversible transcriptional inhibition. The current constructs are small enough to be cloned into AAV. It represents an alternative to doxycycline regulated or rapamyicin-inducible Cas9 systems. Check out the paper here: http://goo.gl/vw5H1Z

A collection of CRISPR tools is available - includes web-based tools for guide RNA design, off-target analysis, NEHJ/HDR analysis, list of plasmids, CRISPR libraries and cloning guides. Probably I still miss a lot, let me know if your favorite resource is not included in the collection. Go tohttp://genedit.blogspot.com/p/new-page.html!

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

Improved off-target prediction by incorporating chromatin data by the CROP-IT algorithm

Off-target cleavage is a big issue in genome editing, particularly with CRISPR. It seems that, although way simpler to use, CRISPR is not as specific as ZNFs or TALENs. A further problem is that predicting off-target sites is very challenging, for example in the recent report of Tsai S et al., an unbiased screening for off-target double strand breaks revealed sites that were not predicted by the MIT CRISPR tool or the E-CRISPR algorithm. The prediction programs however returned several potential sites that did not appear to be off-target sites. More accurate prediction of off-target sites is crucial since most researchers base their guide RNA design of these algorithms. Since for each gene, a huge number of guide RNAs could be potentially designed, accurate prediction of off-target sites would help to score gRNAs based on specificity. At this point we are very far away from this. One major reason for low efficiency prediction is differential accessibility of the DNA depending on chromatin state. The Adli lab (University of Virginia) now developed an improved algorithm for off-target search, the CROP-IT, which incorporates chromatin information.

The CROP-IT allows to search for sites up to 6 mismatches (cleavage) and up to 9 mismatches (binding). This is already an important discrimination, since certain applications (e.g. epigenome editing) requires only Cas9 binding, but not cleavage. Binding or cleavage tolerates different mismatches and now this feature is built into this prediction software. The prediction scores mismatches in relation to the PAM site and then incorporates DN-ase I hypersensitivity information into this score. The PAM site relation scoring is based on training of the algorithm on available ChiP-Seq and GUIDE-Seq data. DN-ase I sensitive sites reflect more open chromatin and more accessible DNA, however this is specific for each and every type of cell. The CROP-IT uses an average from 125 different human cell lines at this point. CROP-IT seems to outperform current prediction algorithms in Cas9 binding and cleavage prediction when validated by ChiP-Seq (Cas9 binding) and cleavage (GUIDE-Seq).

 It is available for S. pyogenes, NGG and NNG PAM sites are analyzed separately.

Link to the paper: http://nar.oxfordjournals.org/content/early/2015/06/01/nar.gkv575.full
Link to the CROP-IT tool: http://cheetah.bioch.virginia.edu/AdliLab/CROP-IT/homepage.html

New Technology based on CRISPR technology: the CRISPR-display.

The John Rinn lab (Harvard, Cambridge, US) utilizes the CRISPR system to bridge RNA cargos onto specific genomic loci enabling the targeting of different RNAs or ribonucleoprotein particles to DNA. The spectacular technology is based on using the dead (inactive) Cas9 /gRNA linked to an RNA of choice (either covalently or by hybridization). Interestingly various RNA cargo could be linked, including natural long-non coding RNAs, aptamers or pools of RNA sequences. This technology has diverse applications, i.e. analysis of lncRNA function, screening for RNA based drugs influencing specific genomic loci, imaging, modular transcriptional regulation.

If you work with lncRNA or RNPs you should check it out!

http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.3433.html

A nice figure showing the rapid evolution of CRISPR research, you can also find out the top 5 patent applicants in the technology! (source: Nature, 'The Disruptor', http://goo.gl/MYEMjR)

2015 American Society of Gene and Cell Therapy Annual meeting in New Orleans, LA - Highlights - GUIDE-Seq reveals a plethora of previously undetected off-target effects with CRISPR

Shengdar Tsai (K. Joung lab, Massachusetts General Hospital, Boston, USA) presented an already published method (http://goo.gl/3pkSxI) to determine off-target effects of CRISPR in an unbiased way. The method is based on the incorporation of a double-stranded oligo by non-homologous end-joining into double-stranded breaks, amplification of the ODN template from genomic DNA and sequencing of the genomic context. This 'genome-wide unbiased identification of double-stranded breaks enabled by sequencing' (GUIDE-Seq) revealed plenty of off-target cleavage with S. pyogenes Cas9. Most of the off-target sites were not predictable by existing biased methods (http://crispr.mit.edu/ and CHiP-Seq), making this method an important addition to the CRISPR toolbox. CRISPR was able to tolerate up to 6 mismatches in the gRNA hybridization, this is much more than previously predicted. Check out the paper for more information.

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.

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

Split-Cas9 design and its implications for AAV-mediated CRISPR delivery

Wright AV et al, PNAS 2015,
www.pnas.org/cgi/doi/10.1073/pnas.1501698112, copyright: PNAS

In their paper, Wright et al. separated the Cas9 enzyme into two distinct parts, a nuclease lobe and an α-helicase lobe. The two different polypeptides were shown to be brought together by the guide RNA, reconstituting the active CRISPR complex. One of the most important applications of this system is that two smaller regions of Cas9 could be cloned into AAV, overcoming capacity limitations of this vector. Moreover, there would be more room for larger or inducible promoters to spatiotemporally limit Cas9 expression. Inducible dimerization domains may also be applied to regulate expression. Readers should be aware that this split Cas9 sytem seems to be less effective than wild-type Cas9, WT Cas9 generated indels in HEK293T cells with around 22% frequency, but this was only 0.6% with split-Cas9 (synchronized cells showed slightly increased indel formation). This warrants further optimization, but having the Cas9 on two different polypeptides definitely has advantages. The lower indel rate compared to WT Cas9 is in accordance with the split Cas9 from the Zetsche B (http://goo.gl/J5Ogqu) paper. The difference in the Wright and the Zetsche paper is that in the latter, the Cas9 is cut in half (to obtain an N- and a C-terminal domain), whereas in the Wright paper the nuclease lobe contains a short N-terminal part and a long C-terminal part (with a short linker in between) and the α-helicase lobe is consisted of 'middle' amino acids. The Wright strategy split Cas9 is able to dimerize spontaneously in the presence of guide RNA, but the Zetsche split Cas9 needs chemically inducible dimerization domains for function.  

Original paper: Wright AV, Sternberg SH, Taylor DW, Staahl BT, Bardales JA, Kornfeld JE, Doudna JA. Rational design of a split-Cas9 enzyme complex. Proc Natl Acad Sci U S A. 2015 Feb 23. pii: 201501698a, http://www.pnas.org/content/early/2015/02/18/1501698112.long

The development of immunological memory in bacteria 

CRISPR mediated bacterial immunity, with permission
from NPG, from Yosef I et al, Nature 2015

Two papers published last week in Nature describe the amazing development of bacterial immunological memory. Previously it has been thought that in contrast to vertebrates, bacteria can not remember previous infections. Now it is obvious that the original function of CRISPR is to fight against invading viruses by cleaving specific parts of the viral genome. During an infection, part of the viral genome is stored in the bacterial genome as a library, from which a matching sequence can be utilized upon subsequent infection. How this happens remained elusive until recently, now it is shown that components of the CRISPR system (Cas1, Cas2, Csn1, Cas9 and tracrRNA) act in concert to find appropriate target regions in the viral genome, and insert a copy into the bacterial genome. Finally it makes sense, why there is a so-called PAM site. This is a very short motif adjacent to the recognition region, which is an absolute sequence requirement of the system (e.g. this is NGG for the S. pyogenes CRISPR system). Most importantly, when pasted into the bacterial genome, there is no PAM site next to the sequence. So that is why bacteria do not attack themselves, since the PAM site is only in the virus, leading to cleavage. That is how bacteria evade autoimmunity. Smart!

Check out the original papers here: http://goo.gl/kz2X1V

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

MPN Genetics Announces Research Grants with focus on CRISPR

Funding option is available for researchers developing genome editing therapies for myeloproliferative diseases. A patient advocacy group called MPN (Myeloproliferative Neoplasms) Genetics and the Leukemia & Lymphoma Society announced "The 2015 MPN Challenge" grant program, one priority area being genome editing in MPN diseases. Deadline is April 1st. Check it out here: http://www.mpnresearchfoundation.org/2015-Request-for-Research-Proposals

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!