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!

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-...

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.

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.

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…..

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).

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).

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.

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.

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.

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.

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.

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).

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.

Zot = best college mascot?

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.