TECHNOLOGY: @nevillesanjana.bsky.social from New York Genome Center and @nyupress.bsky.social discusses the massively-parallel approaches to understand the human noncoding genome and transcriptome at the 2026 AGBT General Meeting.
#AGBTGM26 #FunctionalGenomics #PrecisionMedicine
If you’d like to learn more, please check out all the details in the CRISPore-seq preprint here:
www.biorxiv.org/content/10.1...
Also, our great collaborators at @nanoporetech.com (@sisseljuul.bsky.social #DavidDai & #PriyeshRughani) have been a pleasure to work with on this and other long-read sequencing projects that I hope to tell you about soon. And thanks also to co-authors #WellsBurrell and #ZharkoDaniloski too.
In particular, grateful to Akash for launching the project and Simon for seeing it through to the end and adding some wonderful mechanistic work.
CRISPore-seq would not be possible without a great team. This work was led by talented co-first authors (#SimonMuller, @nathanaelandrews.bsky.social, @rachelyan.bsky.social, #AkashSookdeo) — a set of postdocs & PhD/MD-PhD students from our group.
... and also for training new AI-based virtual cell and DNA language models.
With emphasis recently in academia & biotech on generating massive-scale perturbation single-cell data (e.g. @recursionpharma.bsky.social, #TahoeTx, #XairaTx, and others), full transcript resolution will enable more detailed phenotyping for mechanistic biology...
Overall, CRISPore-seq is exciting given its potential to resolve isoforms for next-generation Perturb-seq.
We also confirmed this with a functional rescue experiment: After CCND1 knockdown, only the isoform containing exon 2 is able to rescue growth (in 2D cell culture and 3D spheroids) and cell cycle progression.
Experimentally, we found that exon 2 of CCND1 is required for binding (via co-IP) of CDK6.
The predicted protein structure using AlphaFold3 shows that exon 2 of CCND1 is important for complex formation with CDK6, a kinase required for cell-cycle progression (G1/S). Exon 2 loss leads to a 2.4-fold decrease in the predicted CCND1/CDK6 protein interface.
One example where we took a deep dive is cyclin D1 (CCND1). Knockdown of the splicing factor SF3B4 doesn’t change overall expression of CCND1 but it changes the transcript (more skipping of exon 2).
So, we combined CRISPore-seq with isoform-specific RNA-targeting CRISPR knockdown data. Here’s how integrating these datasets looks: We see many cases of specific exons that are excluded ✖️❌✖️but are part of essential transcripts‼️.
Given the predominance of exon skipping in CRISPore-seq RBP perturbations, we wanted to understand whether we might find specific transcript isoforms that are important for cell fitness and survival that are being skipped/spliced out.
And, for those RBP’s with binding data (eCLIP from @geneyeo.bsky.social, @darnelr.bsky.social, & #ENCODE), we can see changes in splicing after RBP loss precisely at exons where the RBP binds.
What’s most exciting is that we can deeply catalog 📕📗📘📙📚alternative splicing events after RBP perturbation using the long reads from CRISPore-seq.
We found that exon skipping was the most frequent splicing alteration in our dataset of RBP perturbations. 🦘🦘🦘
The RBP knock-downs result in a clear set of differentially-expressed transcripts and there is a correlation between how essential a RBP is and how many transcripts are disrupted.
Using CRISPore-seq, we decided to perturb several RNA-binding proteins (RBPs) — with different functions and ones that are more or less essential — and map their impact across the long-read transcriptome.
And this is even clearer when we look at individual transcripts and how their exon connectivity changes with different genetic perturbations.
We found that long reads resolved 85% of isoforms, whereas short reads could distinguish only 20%.
We detect 1000s of transcripts in single-cells that would not be possible with short-read — even with many less UMIs per cell.
So, to answer the big question, can we detect more of the diverse isoforms in the human transcriptome?
💯 YES! 🚀🚀🚀
With CRISPore-seq, there is uniform coverage of transcripts from the 5’ end to the 3’ end.
Original ECCITE-seq study (w/ @psmibert.bsky.social): sanjanalab.org/reprints/Mim...
COVID-19 host genes: sanjanalab.org/reprints/Dan...
Noncoding variant discovery: sanjanalab.org/reprints/Mor...
For CRISPore-seq, we used the same 5’ capture strategy (@10xgenomics.bsky.social) as in ECCITE-seq to combine direct CRISPR guide RNA capture with long AND short read sequencing.
To get a transcript-level view of the effect of CRISPR perturbations, we teamed up w/ @sisseljuul.bsky.social & friends @nanoporetech.com to add long-read sequencing to Perturb-seq:
This look at the latest human genome annotation from GENCODE highlights the problem:
There are almost 10-fold more protein-coding transcripts (~200,000) than genes (~20,000).
Looking for some Thanksgiving reading? 🦃🦃🦃
🚨Check out our new preprint on CRISPore-seq!🚨
Combining pooled CRISPR perturbations with single-cell sequencing has been tremendously powerful... but we are missing a lot with current approaches like Perturb-seq and ECCITE-seq.
Thrilled to be a co-organizer (w/ #SidiChen, #CathBollard, #HongboChi) of the first CSHL meeting on Immune Engineering & Cellular Immunotherapy, which kicks off this evening.
Looking forward to a great meeting — and please come introduce yourself if you're attending! #immeng25
⏰Final call! Talk submission for #GenReg26 closes tomorrow, 17 October - don’t miss your chance to be part of the programme.
Check out the initial schedule & explore discounts & grants available👉 bit.ly/4eWDxin
Flyer below with all the key info! #FusionGenomics
Feel free to reach out here (or via email) with any questions.
Although the main conclusion's are unchanged, the preprint contains updates pertaining to individual essential lncRNAs. In parallel, we are working with the journal to correct the scientific record, but we wanted to share the updated work with the community as quickly as possible.
