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Series GSE285778 Query DataSets for GSE285778
Status Public on Jan 03, 2025
Title Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening
Organism Mus musculus
Experiment type Other
Summary Cas12a is a next-generation gene-editing tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene-editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene-editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies.
 
Overall design NGS of mouse cells containing CRISPR/Cas12 libraries (Menuetto/dual and Scherzo/quad). Sequences are of the sgRNA plasmids. Three screens were performed: 1) enrichment screen in mouse EuMyc lymphoma cells using S63845 and Nutlin-3a as selection pressures, 2) drop out screen in immortalised mouse dermal fibroblasts (MDFs), and 3) in vivo enrichment screen for drivers of lymphomagenesis in mice.
 
Contributor(s) La Marca JE, Diepstraten ST, Deng Y, Jin W, Herold M, Fortin J, Haley B
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Submission date Jan 03, 2025
Last update date Jan 03, 2025
Contact name John E La Marca
Organization name Walter and Eliza Hall Institute
Department Blood Cells and Blood Cancer
Street address 1G Royal Parade
City Parkville
State/province VIC
ZIP/Postal code 3052
Country Australia
 
Platforms (2)
GPL16417 Illumina MiSeq (Mus musculus)
GPL30172 NextSeq 2000 (Mus musculus)
Samples (86)
GSM8708881 EuMyc cells, Menuetto, Input, Rep1
GSM8708882 EuMyc cells, Menuetto, Input, Rep2
GSM8708883 EuMyc cells, Menuetto, Input, Rep3
Relations
BioProject PRJNA1206072

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE285778_EuMycCountMenuetto.txt.gz 1.0 Mb (ftp)(http) TXT
GSE285778_EuMycCountScherzo.txt.gz 624.3 Kb (ftp)(http) TXT
GSE285778_InVivoCount.txt.gz 267.1 Kb (ftp)(http) TXT
GSE285778_iMDFCount.txt.gz 1.4 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA

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