Jump to content
World's Largest Herpes Support Group
Sign in to follow this  

CRISPR-CAS 9 New Target Variants

Recommended Posts


For anyone who can't read it:


New CRISPR-Cas9 Variants Improve Targeting Ability of Base Editors

May 20, 2019

NEW YORK (GenomeWeb) – A team led by researchers at the Broad Institute and the University of California, Berkeley has engineered new CRISPR-Cas9 variants that widen the targeting scope of base editors, thus broadening the number of human pathogenic variants that could potentially targeted.

"Indeed, an analysis of human pathogenic single nucleotide polymorphisms in ClinVar reflects a substantial improvement in the fraction of targetable SNPs when considering the expanded [cytosine or adenine base editors'] editing windows compared with their unpermuted counterparts," the authors wrote in their study in Nature Biotechnologytoday


For base editing to successfully occur, the target sequence must be near a protospacer adjacent motif (PAM) that is recognized by the Cas9 domain, and the target nucleotide must be located within the editing window of the base editor. To increase the targeting scope of base editors, the researchers engineered six optimized variants of the adenine base editor (ABE) ABEmax, which use Streptococcus pyogenes Cas9 (SpCas9) variants compatible with non-NGG PAMs.

They also used circularly permuted Cas9 variants (CP-Cas9) to produce four cytosine base editors (CBEs) and four ABEs with an editing window of up to about eight to nine nucleotides, compared to their original editing window of about four to five nucleotides.

"The resulting CP-CBEmax variants exhibit higher product purities, in addition to expanded editing windows, while CP-ABEmax variants maintain the high product purities typical of ABEs," the authors wrote. "These CBE and ABE variants expand the targeting scope of base editing."

The researchers began by creating ABEmax variants that replaced the SpCas9 nickase component with two engineered SpCas9 variants with altered PAM specificities: VRQR-SpCas9 targeting the NGA PAM sequence and VRER-SpCas9 targeting the NGCG PAM sequence. They named these editors VRQR-ABEmax and VRER-ABEmax. They then evaluated the base-editing activity of these ABE variants at six endogenous human genomic loci for each PAM in human HEK293T cells and found that although editing with ABEmax across six endogenous NGA PAM-containing sites resulted in low editing efficiency, editing with VRQR-ABEmax resulted in a 3.2-fold average improvement across all six sites.

They then tested the editing efficiency of ABEmax at six endogenous genomic sites in HEK293T cells containing NGCG PAMs and again observed minimal activity. In contrast, editing with VRER-ABEmax at those sites resulted in a sevenfold improvement over ABEmax.

Further experiments showed that the VRQR-, VRER- and SpCas9-NG variants were compatible with the ABEmax architecture and retained base-editing activity at sites containing their cognate nonNGG PAMs.

To expand the targeting scope of ABE even further, the researchers tried to examine whether Staphylococcus aureus Cas9 (SaCas9) could also be compatible with the ABEmax architecture. SaCas9 naturally targets NNGRRT PAMs, and an evolved variant called SaKKH recognizes NNNRRT PAMs.

The team generated both SaCas9 and SaKKH-ABEmax variants and tested them on six endogenous NNGRRT PAM sites and six endogenous NNHRRT PAM sites in HEK293T cells. They observed moderate editing efficiencies for SaABEmax and SaKKHABEmax, which contrasted with the high activities of SaCas9-derived CBEs that generally edit more efficiently than the corresponding SpCas9 CBE.

"These results suggest that further engineering or evolution may benefit targeting ABE with SaCas9 derivatives," the authors noted.

Given the potential utility of base editors with shifted or expanded activity windows, the researchers next sought to engineer base editor architectures that enabled editing at different protospacer positions. They hypothesized that circularly permuted Cas9 variants might result in expanded or otherwise altered activity windows, and chose five SpCas9 circular permutants — CP1012, CP1028, CP1041, CP1249, and CP1300 — based on both retention of DNA binding activity and predicted proximity to the single-strand DNA loop. They generated five CP-CBEmax and five CP-ABEmax variants and transfected them into HEK293T cells to test their base-editing activity at five endogenous genomic sites containing adenines and cytosines throughout the target 20-nucleotide protospacer.

The researchers found that four of the five CP-CBE variants were capable of base editing at all five sites without substantial indel formation, while CP1300-CBEmax demonstrated highly site-dependent base-editing activity. Three of the top four CP-CBEmax variants exhibited efficient editing activity, and CP1012-CBEmax and CP1028-CBEmax in particular showed broadening of the editing window from the canonical positions four to eight to positions four to 11 of the protospacer.

Similarly, most of the CP-ABEmax variants also exhibited a broadening of the editing window, the researchers said. CP-ABEmax variants retained efficient editing activity similar to that of ABEmax, and both ABEmax and the CP variants generated minimal indels.

Significantly, the researchers found that the window-broadening effect of the CP-ABEmax variants was pronounced, generally resulting in an expansion from the canonical window of protospacer positions four to seven for ABEmax to a window spanning positions four to 12.

When they measured the off-target base-editing efficiency of the CP base editors, the researchers found that it was similar to or less than that of CBEmax or ABEmax for C or A nucleotides within the canonical editing window. For C or A nucleotides outside of the canonical editing window, the expanded editing windows of the CP base editors resulted in higher off-target editing than CBEmax or ABEmax, in some cases.

"Together, these results demonstrate that circularly permuting the Cas9 nickase domain of base editors results in CBEmax and ABEmax variants with broadened or shifted editing windows," the authors concluded.

Share this post

Link to post
Share on other sites

CRISPR hope for vax-resistant disease treatment

Gene-spliced antibodies show promise in mouse trials. Paul Biegler reports.


Gene-editing technology could find solutions where vaccine fail.


Scientists have used the gene-splicing technology CRISPR to make virus-busting antibodies for a range of diseases that cannot, to date, be prevented with vaccines.

The researchers, led by Justin Taylor of the Fred Hutchinson Cancer Research Centre in Seattle, US, say the technique could mean new ways to tackle infections including HIV, influenza and Epstein Barr Virus (EBV), which causes glandular fever.

The team used CRISPR to alter the genetic code of human B-cells, coaxing them to make antibodies against those viruses and another wily agent called Respiratory Syncytial Virus or RSV.

RSV is harmless in healthy people but can be deadly for the very young and old. It causes lung disease that, in the US, puts more than 50,000 kids under five in hospital and kills 14,000 adults over 65 every year.

To see if the rejigged B-cells would actually fight off RSV, Taylor’s team put CRISPR-modified mouse B-cells into healthy mice. The critters were then subjected to a largish dose of RSV up the nostrils. Five days later the researchers took lung samples to see if the virus had taken up residence. RSV was nearly undetectable.

It’s an approach that addresses two problems with the current mainstay in stopping infection before it starts: vaccination. 

Vaccines challenge the body with a small dose of disease to spike immune cells into action. The cells can then be on war footing should the real illness come knocking down the track, helping them beat down the invader. 

But vaccines for some illnesses have been devilishly difficult to make.

The first RSV vaccine trial in 1966 left many infants and toddlers with a severe form of the disease, ultimately killing two. A second trial in 2016 was ineffective. 

HIV is another battlefront, with attempts to make a vaccine underway since the late 1980s.

“Our approach could be used to protect people against infections when a vaccine is not an option,” says Taylor. 

“HIV is a great example of an infection in which a protective vaccine doesn’t exist.”

There is also a whole bunch of people for whom getting vaccinated is out of the question.

These include the sick, elderly and people getting cancer chemotherapy, whose immune system may be so weakened that a vax would simply overwhelm them. Vaccines are a particular threat for people whose immunity has been wiped out in preparation for a bone marrow transplant.

The team’s research therefore included a study that put engineered B-cells into mice that lacked immune cells altogether, mimicking extreme immunodeficiency. After 82 days the cells were still making enough antibody to protect the mice from RSV infection.

It’s a finding that could, ultimately, shift the odds for bone marrow transplant recipients.

“These people are susceptible to a wide variety of viral, bacterial and fungal infections that we could use this technique to protect against while their immune systems recover,” explains Taylor.

The research is published in the journal Science Immunology.

Contribs paulbiegler 2.jpg?ixlib=rails 2.1
PAUL BIEGLER is a philosopher, physician and Adjunct Research Fellow in Bioethics at Monash University. He received the 2012 Australasian Association of Philosophy Media Prize and his book The Ethical Treatment of Depression (MIT Press 2011) won the Australian Museum Eureka Prize for Research in Ethics. 


Share this post

Link to post
Share on other sites

Join the conversation

You can post now and register later. If you have an account, sign in now to post with your account.

Reply to this topic...

×   Pasted as rich text.   Paste as plain text instead

  Only 75 emoji are allowed.

×   Your link has been automatically embedded.   Display as a link instead

×   Your previous content has been restored.   Clear editor

×   You cannot paste images directly. Upload or insert images from URL.

Sign in to follow this  

  • Donate

    If Honeycomb has helped you, please help us by making a donation so we can provide you with even better features and services.

  • The Hive is Thriving!

    • Total Topics
    • Total Posts
  • 0_unsure-if-it-is-herpes.png

    Nervous about dating with herpes? Skip "the talk" and browse profiles here.

  • Posts

    • England1995
      Hi Aussie_gurl, how did you sort this issue out? I am experiencing it.     thanks!
    • MikeHerp
      Hey Guys, I prepared this draft message to the admin at the "+Support Groups" HSV community.  Pls let me know what you think if you have any comments or suggestions before I send it. ________________________________________________ Hello, Thank you for your message.   Perhaps I could run this by you here to get your view first.   In a nut shell, we are part of an HSV community which is helping to support HSV cure research.  I know "cure" is a bit of a dirty word in the HSV community, but please kindly hear me out and, if you don't mind, please check out some of the information I share. The Fred Hutch Cancer Research Center has been making some serious progress towards an HSV cure, even though commercialization is still tentative and some years away.  They are using a technique called gene editing, and with it, they have been able to target the latent form of HSV and destroy it by disrupting its DNA.  They've been able to remove over 50-90% of latent HSV when tested in mice, in effect partially curing mice from HSV. A lot of people are excited about this research.  It is partially funded by the National Institute of Health (NIH), but it is also being supported through private donations from our community. Our donations have enabled their team to progress to testing in guinea pigs before the funding for that was available from the NIH.  I'd like to ask you and the powers that be here, whether it might be ok to post about their research and to also post the link to the donation drive.  Our own admins at Honeycomb.click, have let us post about it and even pinned our main post on this, which has helped us to get many people to contribute to supporting this.  Over 200 members have donated and this has raised $38,000 for this research this year. These funds are being put to good use, as explained in the last video presentation from FHC, where Dr. Jerome who heads up this research has noted that these funds have enabled them to proceed to guinea pig testing. I'd like to share some info links with you. First is a 2016 peer reviewed scientific research paper where they showed they could edit and deactivate 2-4% of latent HSV in mice. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026126/ Next is an article from Mens Health Magazine about the progress of this research from earlier this year, noting that removal of latent HSV has reached up to 90% in mice. https://www.menshealth.com/health/a26306319/herpes-cure-gene-editing/ That article obviously isn't a scientific paper, but it does help to explain what they are doing in layman's terms. Here is The latest from a series of video presentations from the Fred Hutch team.  A little past the 46 minute mark Dr. Jerome gives an acknowledgment to the funds that we have raised and explains how the funding is being put to use.   Finally, a link to the NIH website which shows the grant that this research has received from the NIH last year (this grant will be active through 2023). https://report.nih.gov/categorical_spending_project_listing.aspx?FY=2018&ARRA=N&DCat=Sexually+Transmitted+Infections (Note there are 4 pages there, and the grant for Keith Jerome is on one of the pages only ("Endonuclease-mediated disruption of latent HSV as curative therapy - $450,000 in 2018). Finally, the link to the FHC donations page for supporting this HSV cure research is here:  http://engage.fredhutch.org/site/TR?px=1709211&fr_id=1574&pg=personal After looking at these infos, please kindly consider whether we would be allowed to post about the research and about the donation drive.  We think it is important to get everyone on board, even if it's just a small donation of $10 to $15.  This is now winding through animal studies with the aim of being tested in human clinical trials.  This is a legitimate effort and we think it's an effort that is worth supporting by our community of HSV positives and related stakeholders.  I know you have posted a disclaimer about not posting about "cures", but we presume that to mean fake or scam cures ("the great Dr. Okoloko's medicinal herbal tincture etc.").  This is real and legitimate research and it's exciting. Please kindly consider if you would allow us to share this with your community.  If so, we'd be very happy to share with you first the draft post and take any comments or feedback from you before posting it.   Thank you for your consideration.   
    • sub16610
      Imagine we're both HSV2 positive and we have sex. A few days later I have a huge outbreak. Is it randomness or could having sex with another infected person trigger an outbreak ?
    • vzhe
      400mg Pritelivir has been shown to be worse than a smaller dose daily (and that is what pharmacodynamics would predict as well). You'd expect the same with Amenamevir, though i'm not entirely sure about its half-life right now. TL;DR A weekly dose is for convenience only, i'd always prefer a more effective daily or bi-weekly dose.
    • needahope
      Yup.. Absolutely agreed! 👍 wilson has been helped many of us! 
  • Create New...

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.