Just got my first @plasmidsaurus.bsky.social RNASeq data back and am genuinely blown away🤯 Incredibly easy process, and high-quality data back within 7 days, even from here in the UK!
Header image with the paper title: "Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs"
Make your gene knockouts more efficient with multiplexed Cas12a sgRNAs. Our new paper is out now, with tools available from @addgene.bsky.social , www.plasmids.eu and @vdrc-flies.bsky.social.
www.nature.com/articles/s41...
#CRISPR #geneediting #Drosophila 🧪🧬✂️🔬🪰
Summary 🧵 below.
Hi Jonathan, thank you so much! We think that - since the clonal advantage of the V617F mutation is mild - this re-growth would be very slow. It can take decades from acquisition of the JAK2 mutation to manifestation of clinical disease. But this is something we are keeping an eye on!
Huge thanks to my supervisors @jojdavies.bsky.social Adam Mead + all colleagues @imm.ox.ac.uk who contributed: @yuqi-shen.bsky.social, Zoë Wong, Ramy Slama, Maria Greco, Lai Cheng, @ellouka.bsky.social, Weijiao Zhang, Yavor Bozhilov, @jhamley98.bsky.social, Adam Wilkinson, @bethpsaila.bsky.social
We are excited about this data, since the decade-long progression of V617F MPNs means that – unlike for aggressive malignancy – a single genome editing intervention may provide meaningful long-term suppression of the mutant clone. Watch this space, especially as in vivo editing techniques mature!
Collectively, our data provides evidence that JAK2 V617F does not cause oncogene addiction, and that targeting the mutant copy reverts heterozygous cells to a more normal phenotype.
Intriguingly, cells with highly edited V617F copies can be detected at the experimental endpoint in the treatment group. This shows that editing V617F drastically reduces aggressiveness but preserves engraftment potential (presumably supported by the wildtype copy of JAK2 in SET-2 cells).
Finally, we undertook xenograft experiments. Targeting the V617F mutation in SET-2 cells prior to transplantation results in dramatically improved survival and fully reverted splenomegaly and bone marrow fibrosis phenotypes.
Transcriptionally, inactivation of V617F reverts inflammatory gene expression signatures caused by the mutation (more details in the preprint!).
Since editing creates many genotypes (edited/unedited for heterozygous/homozygous cells each), we used combined single-cell RNAseq and genotyping (TARGET-Seq) to resolve effects by genotype. Again, we see remarkable efficiency: 97% of mutant cells have all mutant copies inactivated by CRISPR.
To further validate that edited CD34+ cells retain normal haematopoiesis, we engrafted these cells into a bone marrow organoid system. We saw normal multilineage myeloid differentiation and deep and persistent drops in VAF – as much as 99% down from the baseline burden!
Editing preserves colony-forming ability while reducing erythroid output. Intriguingly, by genotyping individual colonies, we find that heterozygous mutant cells tolerate loss of their mutant alleles (MUTx).
After testing several strategies, we find that a Cas12 Ultra approach - which exploits a de novo PAM created by the V617F mutation - yields the best results. We achieve >97% V617F inactivation in primary patient CD34+ cells via electroporation, with minimal targeting of wildtype alleles!
Since MPNs originate from stem cells, it should be possible to genetically delete V617F alleles from CD34+ cells. If we can do this allele-selectively (AS) for the V617F alleles, we should selectively suppress mutant effects while preserving haematopoiesis from wildtype alleles.
JAK2 V617F is a common haematological driver mutation, but JAK2 inhibitors fail to clear mutant cells in most patients with myeloproliferative neoplasms (MPNs).
Excited to share that a part of my PhD work is online now on BioRxiv!
www.biorxiv.org/content/10.6...
We developed a genome editing approach to target the JAK2 V617F mutation and demonstrate its potential to alleviate MPN hallmarks in primary patient cells.
Thread 👇
Check out the latest work from the @davieslab.bsky.social led by Hangpeng!
Enhancer scrambling strategy
We are happy to share our enhancer scramble story, a strategy to create hundreds of stochastic deletions, inversions, and duplications within mammalian gene regulatory regions and associate these new architectures with gene expression levels 🧵
www.biorxiv.org/content/10.1...
Our new preprint is out - please share!
We asked: How can we make CRISPR knockouts in vivo more efficient? And set up an assay that enables detection of CRISPR cutting with 100s-1000s of sgRNAs over entire chromosome arms in living animals.
Grab a ☕ and let’s dive in. 🧵🧪 1/n
#CRISPR #geneediting
Feels like the migration of science twitter is actually working this time? Exciting!
Some quite interesting observations on length bias of different PCR polymerases, from www.nature.com/articles/s41...
Maybe I need to reconsider my unconditional love for Q5...
