TY  - JOUR
TI  - Engineering a CRISPRoff Platform to Modulate Expression of Myeloid Cell Leukemia (MCL-1) in Committed Oligodendrocyte Neural Precursor Cells
AU  - Gil, Melanie 
AU  - Hamann, Catherine Ann
AU  - Brunger, Jonathan M. 
AU  - Gama, Vivian 
VL  - 14
IS  - 1
PY  - 2024
DA  - 2024/01/05
SP  - e4913
C1  - Bio-protocol 2024;14:e4913
DO  - 10.21769/BioProtoc.4913
UR  - https://doi.org/10.21769/BioProtoc.4913
AB  - In vitro differentiation of human pluripotent stem cell (hPSC) model systems has furthered our understanding of human development. Techniques used to elucidate gene function during early development have encountered technical challenges, especially when targeting embryonic lethal genes. The introduction of CRISPRoff by Nuñez and collaborators provides an opportunity to heritably silence genes during long-term differentiation. We modified CRISPRoff and sgRNA Sleeping Beauty transposon vectors that depend on tetracycline-controlled transcriptional activation to silence the expression of embryonic lethal genes at different stages of differentiation in a stable manner. We provide instructions on how to generate sgRNA transposon vectors that can be used in combination with our CRISPRoff transposon vector and a stable hPSC line. We validate the use of this tool by silencing MCL-1, an anti-apoptotic protein, which results in pre-implantation embryonic lethality in mice; this protein is necessary for oligodendrocyte and hematopoietic stem cell development and is required for the in vitro survival of hPSCs. In this protocol, we use an adapted version of the differentiation protocol published by Douvaras and Fossati (2015) to generate oligodendrocyte lineage cells from human embryonic stem cells (hESCs). After introduction of the CRISPRoff and sgRNAs transposon vectors in hESCs, we silence MCL-1 in committed oligodendrocyte neural precursor cells and describe methods to measure its expression. With the methods described here, users can design sgRNA transposon vectors targeting MCL-1 or other essential genes of interest to study human oligodendrocyte development or other differentiation protocols that use hPSC model systems.Key features• Generation of an inducible CRISPRoff Sleeping Beauty transposon system.• Experiments performed in vitro for generation of inducible CRISPRoff pluripotent stem cell line amenable to oligodendrocyte differentiation.• Strategy to downregulate an essential gene at different stages of oligodendrocyte development.Graphical overviewWorkflow for generating inducible CRISPRoff stem cell line and assessing knockdown phenotype in stem cell–derived committed oligodendrocyte neural precursor cells
KW  - Oligodendrocytes
KW  - Oligodendrocyte precursor cells
KW  - CRISPR/Cas
KW  - Sleeping Beauty
KW  - MCL-1
KW  - Pluripotent stem cells
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False