Neville Sanjana
@nevillesanjana.bsky.social
Scientist at the New York Genome Center & NYU.
http://sanjanalab.org
http://sanjanalab.org
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.
November 27, 2025 at 11:59 AM
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.
November 27, 2025 at 11:59 AM
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.
November 27, 2025 at 11:59 AM
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).
November 27, 2025 at 11:59 AM
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‼️.
November 27, 2025 at 11:59 AM
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‼️.
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.
November 27, 2025 at 11:59 AM
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. 🦘🦘🦘
We found that exon skipping was the most frequent splicing alteration in our dataset of RBP perturbations. 🦘🦘🦘
November 27, 2025 at 11:59 AM
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. 🦘🦘🦘
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.
November 27, 2025 at 11:59 AM
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.
November 27, 2025 at 11:59 AM
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 found that long reads resolved 85% of isoforms, whereas short reads could distinguish only 20%.
November 27, 2025 at 11:59 AM
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 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.
November 27, 2025 at 11:40 AM
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.
💯 YES! 🚀🚀🚀
With CRISPore-seq, there is uniform coverage of transcripts from the 5’ end to the 3’ end.
November 27, 2025 at 11:40 AM
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.
💯 YES! 🚀🚀🚀
With CRISPore-seq, there is uniform coverage of transcripts from the 5’ end to the 3’ end.
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.
November 27, 2025 at 11:40 AM
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:
November 27, 2025 at 11:40 AM
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).
There are almost 10-fold more protein-coding transcripts (~200,000) than genes (~20,000).
November 27, 2025 at 11:40 AM
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).
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.
🚨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.
November 27, 2025 at 11:40 AM
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.
🚨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.
Getting back to the MYC story, we next wondered whether the CREs we identified bind specific TFs. We looked for those TFs whose expression was correlated with MYC expression and where binding sites could be identified in the CREs.
March 3, 2025 at 2:57 AM
Getting back to the MYC story, we next wondered whether the CREs we identified bind specific TFs. We looked for those TFs whose expression was correlated with MYC expression and where binding sites could be identified in the CREs.
There is likely a combination of effects on growth driven by both a genomic enhancer and ncRNA transcript since expressing the same ncRNA in trans partially (but not completely) rescues the phenotype!
March 3, 2025 at 2:57 AM
There is likely a combination of effects on growth driven by both a genomic enhancer and ncRNA transcript since expressing the same ncRNA in trans partially (but not completely) rescues the phenotype!
Although CCAT1-MYC looping was the largest change upon CCAT1 transcript loss, this wasn’t just a local change: Many DNA contacts were impacted outside of the MYC TAD — on the same chromosome and genome-wide!
March 3, 2025 at 2:57 AM
Although CCAT1-MYC looping was the largest change upon CCAT1 transcript loss, this wasn’t just a local change: Many DNA contacts were impacted outside of the MYC TAD — on the same chromosome and genome-wide!
How could a lncRNA impact gene expression?
We found that loss of the CCAT1 transcript led to DNA conformation changes; the physical contact between the DNA was impacted by the RNA.
We found that loss of the CCAT1 transcript led to DNA conformation changes; the physical contact between the DNA was impacted by the RNA.
March 3, 2025 at 2:57 AM
How could a lncRNA impact gene expression?
We found that loss of the CCAT1 transcript led to DNA conformation changes; the physical contact between the DNA was impacted by the RNA.
We found that loss of the CCAT1 transcript led to DNA conformation changes; the physical contact between the DNA was impacted by the RNA.
One of the CREs identified in 3 cell lines is located at the promoter for the lncRNA CCAT1 (we can’t escape lncRNAs these days...!)
We found that cell growth decreased when the DNA was silenced AND when the CCAT1 transcript was targeted using a RNA-targeting CRISPR (Cas13).
We found that cell growth decreased when the DNA was silenced AND when the CCAT1 transcript was targeted using a RNA-targeting CRISPR (Cas13).
March 3, 2025 at 2:43 AM
One of the CREs identified in 3 cell lines is located at the promoter for the lncRNA CCAT1 (we can’t escape lncRNAs these days...!)
We found that cell growth decreased when the DNA was silenced AND when the CCAT1 transcript was targeted using a RNA-targeting CRISPR (Cas13).
We found that cell growth decreased when the DNA was silenced AND when the CCAT1 transcript was targeted using a RNA-targeting CRISPR (Cas13).
How are these CREs regulating MYC?
Using H3K27ac HiChIP we found that all but one of the CREs is in physical contact with the MYC promoter. ➰
However, contact 👉👈 is not enough for regulation: less than 10% of all contacts to the MYC promoter were identified as CREs.
Using H3K27ac HiChIP we found that all but one of the CREs is in physical contact with the MYC promoter. ➰
However, contact 👉👈 is not enough for regulation: less than 10% of all contacts to the MYC promoter were identified as CREs.
March 3, 2025 at 2:43 AM
How are these CREs regulating MYC?
Using H3K27ac HiChIP we found that all but one of the CREs is in physical contact with the MYC promoter. ➰
However, contact 👉👈 is not enough for regulation: less than 10% of all contacts to the MYC promoter were identified as CREs.
Using H3K27ac HiChIP we found that all but one of the CREs is in physical contact with the MYC promoter. ➰
However, contact 👉👈 is not enough for regulation: less than 10% of all contacts to the MYC promoter were identified as CREs.
We perturbed these CREs in 1 or 2 cell lines were the CRE is present (from the CRISPRi screens) and 1 where it wasn’t. Indeed, we see decreased cell growth and decreased MYC expression in the cell lines with the CRE and no change in cells lines without the CRE.
March 3, 2025 at 2:43 AM
We perturbed these CREs in 1 or 2 cell lines were the CRE is present (from the CRISPRi screens) and 1 where it wasn’t. Indeed, we see decreased cell growth and decreased MYC expression in the cell lines with the CRE and no change in cells lines without the CRE.
Even for CREs that were previously identified in human or mouse studies (mostly using large deletions), we were able to increase the resolution of the CRE boundaries. 🎛️🗺️ Up to 10,000-fold in one case!
March 3, 2025 at 2:43 AM
Even for CREs that were previously identified in human or mouse studies (mostly using large deletions), we were able to increase the resolution of the CRE boundaries. 🎛️🗺️ Up to 10,000-fold in one case!
We identified 32 cis-regulatory elements (CREs) across the cancer types — many of which were new ones. Most of these CREs are unique to a cancer/cell type, but some are shared.
March 3, 2025 at 2:43 AM
We identified 32 cis-regulatory elements (CREs) across the cancer types — many of which were new ones. Most of these CREs are unique to a cancer/cell type, but some are shared.