iiinova — where your digital self keeps evolving

Haley Lab @labohaley.bsky.social · 21/03/2025

Exciting week for the lab. We fired up our @elembio.bsky.social Aviti for its inaugural run (a success!), fully enabling several ongoing CRISPR screens. Thx @gabsflors.bsky.social, Xiaozhen Wen, David Sharon, Pierre Chagnon, and members across our teams for helping to onboard the new sequencer!

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Haley Lab @labohaley.bsky.social · 16/03/2025

Our institute (affiliated with @umontreal.ca) is hiring! Come be our colleague and start your independent research career in the wonderful, science-friendly city of Montreal. See images for more details. You can find info about CR-HMR via this link: crhmr.ciusss-estmtl.gouv.qc.ca/en/about-cr-...

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Haley Lab @labohaley.bsky.social · 28/12/2024

Lastly, for users who would like to avoid Cas9-induced dsDNA breaks, we’ve established a leak-free, fully-inducible version of @miketilapia.bsky.social's ZIM3-dCas9 vector – which showed dramatically greater silencing compared to the older KOX1-based KRAB-dCas9 system.

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Haley Lab @labohaley.bsky.social · 28/12/2024

pUltra-tight relies on dox-induced expression of Cas9. But, dox can be toxic to some cells, or maybe you want to reserve dox for induction of a different transgene. So, we incorporated an improved “Xon” splice switch module that will active Cas9 expression in the presence of branaplam.

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Haley Lab @labohaley.bsky.social · 28/12/2024

One application that particularly excited us was deletion of genomic segments (disrupting non-standard targets). Flank a target with two guide RNAs, induce, & the intervening segment is removed. We deleted a 2kb chunk of H3-3A, but you can also use this for enhancers, miRNAs, specific exons…..

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Haley Lab @labohaley.bsky.social · 28/12/2024

While many of the modules helped, inclusion of a new, degron-linked anti-CRISPR proved critical, resulting in "pUltra-tight" (pTET-DD-Cas9;AcrIIA4-LID). We show that that pUltra-tight works as expected across multiple cell lines and target genes (shown below for CD81 KO in 293T cells).

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Haley Lab @labohaley.bsky.social · 28/12/2024

To tackle this in a comprehensive manner, we incorporated a variety of distinct Cas9 activity control modules, 1x1 and in combinations, using an all-in-one piggyBac transgenic vector that includes a constitutive guide RNA-expression cassette (or a multi guide cassette).

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Haley Lab @labohaley.bsky.social · 28/12/2024

Like many others, we recognized that temporal control of Cas9 activity opened the door to a ton of interesting experiments. But, the “vanilla” inducible Cas9 system (TRE3G-Cas9, doxycycline induction) was super leaky, with >60% editing of a given target before induction.

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Haley Lab @labohaley.bsky.social · 21/12/2024

Using an improved ASO splice-switch reporter + CRISPR-KO screening, the team identified AP1M1 as a key negative regulator of this mechanism. Many more details in the text, where the team extends findings from the in vitro screens to in vivo validation.

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Haley Lab @labohaley.bsky.social · 09/12/2024

And, that worked out every bit as hoped! Shown here is one example of a known phenotype (KDM1a/LSD1 depletion = Vim and/or HLA-I upregulation). More examples in the main text and supplement.

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Haley Lab @labohaley.bsky.social · 09/12/2024

With these bits in place, Valentina executed a focused screen, looking at the loss-of-function phenotypes for ~50 highly-expressed epigenetic modifier genes in a cancer cell line, based on a hypothesis that targeting these would lead to distinct transcriptional changes.

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Haley Lab @labohaley.bsky.social · 09/12/2024

Valentina adapted elements of the CROP-seq vector concept to work some magic with our guide RNA cassette, optimizing for single cell Cas12a RNA library expression/prep, whether delivering 1, 2, or even 4 guide RNAs in tandem.

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Haley Lab @labohaley.bsky.social · 09/12/2024

Expressing FKBP-enAsCas12a w/a guide library (to KO targets) then depleting the enzyme for ~48 hrs via dTag, guide RNA transcripts/barcodes accumulate & can be used to link each guide to a phenotype by scRNA-seq. Results w/depleted enAsCas12a look similar to cells expressing guides only (no enzyme).

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Haley Lab @labohaley.bsky.social · 09/12/2024

Long-story short, Cas12a chews up the transcripts used to assign guides (crRNAs) to individual cells. Taking cues from several studies (via @bkleinstiver.bsky.social , @johndoench.bsky.social, Grey, Bock, and more), we adapted enAsCas12a with a dTag-sensitive degron module.

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Haley Lab @labohaley.bsky.social · 10/09/2023

On Aug 1, 2023 we opened the doors to our new lab at the Université de Montréal and Centre de recherche de l'Hôpital Maisonneuve-Rosemont. Funded positions open for trainees & staff interested in functional genomics, genome engineering, and molecular genetic tech dev.

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