7 cool recent CRISPR articles

CRISPR Model Jacob Corn

CRISPR Model from Jacob Corn

So everyone is buzzing about the CRISPR patent court decision (which BTW I think was flawed but that’s for another post), but the research roars on at warp speed.

Here are 7 recent CRISPR articles that caught my attention.

What are your favorite recent CRISPR papers?

Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration. Do you think the term “genome surgery” is appropriate?

Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells. CRISPR on the brain.

Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. CRISPR pre-clinical promise.

The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia. CRISPR vs. cancer.

Expanding the CRISPR Toolbox: Targeting RNA with Cas13b. CRISPR systems continue to evolve.

CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells. CRISPR’ing ES cells.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells. I blogged on this one here and did an opinion piece at WaPo here.

The Niche top posts of 2016

stem cell fireworksWhat were the top posts here on The Niche for the past year? I’ve listed some of them below along with some posts from 2015 that remain highly read.

Some top 2016 posts

2015 and older posts that remain highly read every day

Interview with Fredrik Lanner who is CRISPR’ing healthy human embryos

In the past year there has been a great deal of attention given to the potential use of CRISPR-Cas9 for gene editing in human embryos. An important recent development, described in a new NPR article by Rob Stein, is the use of CRISPR-Cas9 in healthy human embryos for developmental biology research by assistant professor Fredrik Lanner of The Karolinska Institute. Dr. Lanner, who invited Stein into his lab to observe the work, kindly agreed to do a Q&A interview with me (below) on his team’s use of CRISPR-Cas9 gene editing for research in healthy human embryos.
ssistant Professor Fredrik Lanner

Assistant Professor Fredrik Lanner. Picture by Rob Stein

PK: What got you interested in doing gene editing in healthy human embryos?

FL: I studied mouse preimplantation development during my postdoc in Janet Rossant’s lab and one of the discoveries we made was the importance of fgf-erk signaling in EPI-PE segregation (Yamanaka, Lanner and Rossant Development 2010). A couple of years later two papers showed that the same mechanism is not controlling the same segregation in human embryos. Since then it has become widely appreciated that the mouse probably is not such a great model system for the human and we really need to start studying human embryos to understand human preimplantation development. I therefore moved back to Sweden, Karolinska Institutet to start my own lab with that specific focus. As a first start we have built a transcriptional single cell roadmap of how the first cell types emerge during the first week of human development (Petropoulos et al Cell 2016). We now want to move from descriptive to functional studies. For this we are of course using pharmacological inhibitors for various signaling pathways but to be able to elucidate which transcription factors are important for how the first cell types are established and how pluripotency is controlled we need other approaches. CRISPR is therefore an obvious next step to evaluate.

PK: Did you have to get some kind of official approvals from your own Karolinska Institute? Did you also need some kind of approval from the Swedish government?

FL: We applied for and got ethical permits from the Swedish regional ethics board (EPN.SE) last spring, 2015. We have also lifted these experiments in KI’s internal ethics board, to inform the KI leadership of our plans and to make sure we had their support.

The Swedish law is clear that genome editing is only allowed within the first 14 day as long as the embryo is not transferred back for a continued pregnancy. This means that heritable genome editing for clinical purposes would not be allowed in Sweden. The clear legislation has been key in us moving ahead with these plans.

PK: What is the source of funding for this work?

FL: Towards the functional gene studies I have internal funding from KI and external funding from the Knut and Alice Wallenberg foundation and through Lau fellowship. For our embryo research I also have funding from the Swedish Research Council, Ragnar Söderberg fellowship and the Swedish Strategic Research Foundation.

PK: Did you receive any kind of bioethics training related to CRISPR’ing human embryos or discuss it with a bioethicist before beginning?

FL:  We have discussed it within the KI ethics council consisting of people with legal, ethics and research expertise. I have further presented and discussed at the symposium organized by National Academies of Sciences in Paris http://www.nationalacademies.org/gene-editing, and a Scandinavian meeting organized by The Norwegian Biotechnology Advisory Board. Early October I will discuss this further with The Swedish Gene Technology Advisory Board. We have followed these discussions closely during the last two years.

PK: I realize you declined to say to Rob Stein what gene(s) you are targeting, but can you name them now? My own view is that with gene editing of human embryos that transparency is needed combined with a strong base rationale, which together make for good reasons to be open publicly about the genes being targeted. If you can’t say the genes is it because you’re concerned about competition from other researchers?

FL:  We are targeting genes that we think will be involved in lineage specification and establishing pluripotency. We want to be open but I’m still not ready to disclose exactly which genes we will focus on.

PK: Are you aware of other teams in your own or other countries doing gene editing in healthy human embryos? I’m trying to get a sense of how much of this kind of work is ongoing around the world.

FL:  No I only know of Kathy Niakans’ plans to look at similar questions.

PK: Is one of your ultimate goals to aid in fertility treatments? Would this involve in the future germline gene editing of human embryos then used to make people if all went well? Or would it rather be based on the knowledge you gain, but applied in a non-gene editing approach during reproduction? How are you seeing this play out in the future?

FL:  We are trying to generate fundamental knowledge and we don’t have any ambition to move in that direction. I’m actually pretty skeptical that the technology will be used for genome editing in the early embryo anytime soon. My questions concerns efficiency, safety and competitiveness compared to preimplantation genetic diagnosis. Targeting somatic cells is already leading the development of this technology.

PK: What is the source of the human embryos being used in your research?

FL:  The embryos are from infertility treatments where the couples mostly have gotten their children. In Sweden you can only store the embryos frozen up to 5 years after which they will be destroyed. At that point they can instead donate the embryos to research. These embryos are frozen at embryonic day 2 at which time the embryo consists of 4 cells.

PK: How did you decide to invite a journalist into you lab to observe the work?

FL:  Since NPR has a good reputation I did not hesitate to let Rob Stein come and visit if he could come the date we were planning to perform the experiment and as long as it did not impact on the practical work.  However, it is clear that we can not have reporters in the lab while we perform experiments on a regular basis.

PK: Anything else you feel is important to know?

FL: I would like to emphasize that we have not rushed into this but spent extensive time evaluating targeting strategy in human ES cells. We got the ethical permit during the spring of 2015 after which we have followed and participated in national and international discussions surrounding this technology over a year. This discussion has led to several organizations recommending that the fundamental research in cultured embryos is acceptable and important whereas the clinical translation of the technology with intention to generate a person is not. This in accordance to our Swedish legislation and has encouraged us to initiate these studies to evaluate the feasibility to study gene function in early human embryos using CRISPR-Cas9. I would also like to emphasize that I strongly think these experiments should be performed in genetically normal embryos if we are to learn anything about normal human preimplantation development.

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Nature Biotechnology looking at NgAgo paper amidst reproducibility concerns

When potentially game changing new technologies are reported such as NgAgo gene editing, both scientists and the public get excited, but especially if such new reports stem from a single paper it is wise to take a cautious approach for a while. The key question is whether the new findings will turn out to be reproducible.

With the case of NgAgo specifically, the Nature Biotechnology paper reporting potentially very desirable gene editing properties, drew a lot of interest. See archived blog posts on NgAgo here.

NgAgo China newspaper

Snapshot of part of China Daily article. Photo Credit Dr. Robert Geller

However, recently many within the scientific community have reported consistent difficulties in getting NgAgo to function as reported. Gaetan Burgio did a guest post here presenting 7 figures of data that together paint a picture of NgAgo not functioning at all like CRISPR.

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Gaetan Burgio New Data & Theory on NgAgo versus CRISPR

By Gaetan Burgio

CRISPR/Cas9 genome editing has dramatically changed our way to perform biological experiments. While highly efficient and easy to use, one limitation with CRISPR/Cas9 mediated genome editing technology is the occurrence of off-target effects and the restriction of the PAM recognition sequence. Many modifications from the original system have been proposed to improve its efficiency, specificity and to avoid off-target effects. Recently a new system based on the bacteria Natronobacterium gregory Argonaute (NgAgo) was proposed as a serious alternative to CRISPR/Cas9. NgAgo is based on a DNA recognition pattern and unlike all the systems based on CRISPR doesn’t require a PAM recognition sequence. The target specificity is mediated from a phosphorylated oligonucleotide on the 5′ end. As it doesn’t require any cloning or in-vitro transcription, it was sought to be a serious alternative to the CRISPR-Cas9 system.

Recently an astonishing paper published in Nature Biotechnology from Chunyu Han’s group in China proposed NgAgo as a simple system to edit cell lines. As many, I was particularly interested to establish the protocol in my laboratory. The recent availability of the plasmid from Addgene encouraged us to establish this protocol and this is what I tried to do in the last two months or so. Below is a summary of my experience with NgAgo.

Reproducing Han’s paper results:

Firstly I decided to not repeat Han’s experiment stricto sensu as my group works primarily on mouse zygotes. The sequences targeted in this paper were all specific to the human genome. Instead I’ve chosen a gene that I’ve been working on for a very long time (Beta-spectrin) and used it to make my first CRISPR/Cas9 edited mouse line over 2 and 1/2 years ago. Usually to establish a new technique, I use a set of highly efficient sgRNA targeting this gene. These sgRNA are working extremely well and are extremely helpful to improve the technology in my hands.

We had a first attempts on Beta-spectrin gene by co-injecting the NLS-NgAgo-GK plasmid at 5 ng/µl with various concentrations of 5′ phosphorylated oligo (2.5, 25 and 50 ng/µl) purchased from IDT into the mouse zygote. After co-injection of the mix into the pronucleus, we cultured the zygotes for 4 days to blastocyst stage and extracted the DNA for PCR and Sanger sequencing.

Many extra bands on the gel electrophoresis:

The first results from our PCR are below (Figure 1) and were very exiting for us. It showed many extra-bands on the gel. I thought these were products of the edited genome as I see often with CRISPR/Cas9. At that time I was at the TAGC conference in Orlando, USA. I showed the results to my colleagues and after few discussions with them I decided to release this gel picture below (Figure 1) from my twitter account.

Burgio Figure 1

Figure 1: PCR on mouse Blastocysts after NgAgo Pronuclear injection in zygotes
We then performed the T7 endonuclease assay on these PCR products (Figure 2) and surprisingly we couldn’t see a clear difference with the original PCR, which was very strange.

We then performed the T7 endonuclease assay on these PCR products (Figure 2) and surprisingly we couldn’t see a clear difference with the original PCR, which was very strange.

Figure 2: T7E treatment on the PCR product of the NgAgo injected Zygotes Interestingly at higher concentration of 5' phosphorylated oligo and the same primer set, these extra bands almost disappeared (see Figure 3). We saw this with others genes too (Tet1 and Tet2).

Figure 2: T7E treatment on the PCR product of the NgAgo injected Zygotes
Interestingly at higher concentration of 5′ phosphorylated oligo and the same primer set, these extra bands almost disappeared (see Figure 3). We saw this with others genes too (Tet1 and Tet2).

Interestingly at higher concentration of 5′ phosphorylated oligo and the same primer set, these extra bands almost disappeared (see Figure 3). We saw this with others genes too (Tet1 and Tet2).

Burgio Figure 3

Figure 3: PCR and electrophoresis gel on mouse Blastocysts after NgAgo Pronuclear injection in zygotes with high concentration of 5′ Phosphorylated oligo

Meanwhile I discovered from many discussions on my Twitter account, at the TAGC meeting, emails I have received and from this interesting Google group discussion thread that many have tried to replicate Han’s results using his experimental setup, in human cell lines, mouse or zebrafish with NgAgo DNA, mRNA or protein. They all failed to edit the genome.

First Sanger sequencing results:

We then performed a first round of Sanger sequencing and the chromatograms were an absolute mess (Figure 4) to a point that we couldn’t properly identified any sequences (Except from the wild type allele) as many alleles were amplified. However, by matching the guide to the sequences, I had the suspicion that 2 samples were edited (from Figure 1, samples 3 and 8)

Burgio Figure 4

Figure 4: Typical Sanger sequencing run we had from NgAgo, 5′ phosphorylated oligo injection into zygotes, culture to Blastocyst, DNA extraction, PCR amplification and sequencing.

Second Sanger sequencing results:

We then performed again the PCRs and decided to cut every single extra band from the electrophoresis gel and send those to Sanger sequencing to determine whether these were sequences from the plasmid, from the edited beta-spectrin gene or primer dimers. Couple of discussions I had on twitter or elsewhere mentioned that the 5′ Phosphorylated oligo could act as a primer and amplify the genome, which is possible and I will come back to this later. The results are in Figure 5 and show convincingly that these extra bands were the amplification of random sequences.

Burgio Figure 5

Figure 5: Electrophoresis gel of mouse zygotes micro-injected with NgAgo

I must make 2 important comments: 1) The primers are specific to the sequence of interest. we have performed tons of PCRs using this primer set and we never saw these extra bands. 2) This result is specific to the low 5′ phosphorylated oligo concentration setup and is almost nonexistent with 25 ng/µl of 5′ phosphorylated oligo.

Clustal alignment:

Initially I thought these sequences were random but I wasn’t quite sure. To test this hypothesis, I aligned all these sequences together using Clustal to see whether I could identify a common pattern. The results are presented in Figure 6 using the results from Sanger sequencing (forward primers). The results are similar for the Reverse primers and I won’t show it here.

Burgio Figure 6

Figure 6: Typical Clustal alignment of all the sequences cut from the electrophoresis gel. The Ank-1 is the reference sequence.

There is clearly a common pattern which doesn’t match at all the 5′ phosphorylated oligo. However it matches with the sequences from the Forward and Reverse primers but quite imperfectly and I will come back to this later. The first hypothesis that came into my mind is my primers are not specific enough. Although it didn’t explain 1) Why at 25 ng/µl of 5′ Phosphorylated oligo I don’t see this pattern, 2) I should have for a long time noticed this given I have genotyped and Sanger sequenced over 100 CRISPR/Cas9 edited mice using these primers and 3) the initial PCRs (Figure 1,2 and 3) showed no extra-bands for the B6 (C57BL/6) DNA control or the water.

To investigate this further, I hypothesised that a foreign DNA sequence (plasmid or other nucleotides from the mouse genome) integrated to these amplified sequences. To test this, I Blast searched the sequences to the mouse genome and the primer pairs for each sequence that were cut from the gel. One example is presented in Figure 7. I found the same pattern for the Forward and Reverse primers for all samples that I have tested.

Burgio Figure 7

Figure 7: chromatogram of one typical sequence (here Reverse primer)

Figure 7 shows two features. Firstly the first 6 to 9 nucleotides from the Forward and the Reverse primers match perfectly with the endogenous sequence. Secondly the remaining 13 to 16 nucleotides from the primer pairs were added to the endogenous sequence. This explains the amplification of these extra bands on the gel (Figure 1). This primer pair was not phosphorylated and no ligase was added to the PCR and sequencing reactions.

NgAgo: A ligase enzyme?

From these results, my hypothesis is as following: The NgAgo plasmid was injected into the zygotes and NgAgo was transcribed and translated into a protein, possibly at zygote stage. The enzyme certainly persisted to blastocyst stage at 37ºC and remained intact after DNA isolation from the blastocysts. The PCR reaction certainly activated the NgAgo enzyme, which functioned as ‘a ligase’ under the classical PCR conditions and added the 10 to 15 nucleotides to the endogenous sequences that were matched with the first 6 to 10 nucleotides of the primer pairs. Interestingly this ‘ligase’ activity from NgAgo seems to be inhibited at high concentration of 5′ Phosphorylated oligo. My hypothesis is this might have degraded the NgAgo enzyme.

My Hypothesis on how NgAgo function:

After these series of experiments, these are my thoughts on NgAgo. Firstly, as many elsewhere found, I have found strictly NO EVIDENCE for a genome editing with NgAgo after multiple attempts with various settings and 3 different genes. Secondly I found instead a ‘Ligase’ like activity of NgAgo under normal PCR conditions, which has strictly nothing to do with the endonuclease activity claimed in Han’s paper. It seems to me that the NgAgo enzyme needs to be heated over 50ºC to function, which is in direct contradiction to the Han’s paper.

My take on all these failed experiments trying to reproduce Han’s paper is basically the incubation temperature of the cells is too low for the enzyme to function or the enzyme/5′ phosphorylated oligo complex is rapidly degraded within the cells explaining possibly why nobody has been able to reproduce Han’s experiment. NgAgo may or may not have an endonuclease activity creating a double strand break but under so specific conditions that they are almost impossible to reproduce and too restrictive for a broad use of this system if this is real. Additionally I do have some serious doubt on NgAgo over its endonuclease activity. Nature Biotechnology should ask Han to release all his raw data + experimental condition to the public. This is a duty of care from the journal. Finally I do believe strongly that whatever happens with NgAgo. the CRISPR/Cas9 system will be there for a very long time and NgAgo will be rapidly abandoned after such failed attempts from everyone in the genome editing field. There is clearly no bright future for NgAgo.

My view on Open Science:

Finally I would like to conclude my post by acknowledging all the people in my laboratory, on Twitter and elsewhere that have contributed to this story. It was my first open science experience and I found the discussion with my peers highly stimulating. I think rather than to chase high impact publications and be secretive, we should be more open and share our results to avoid everyone wasting their time on results that are irreproducible and pointless. In my opinion this is the way Science should work.

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