Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However, the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific single-guide RNAs (sgRNAs), the induced dCas9-KRAB efficiently maintains gene inactivation, although it modestly down-regulates the expression of active genes. The proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knock-in (KI) mouse model that enables the shutdown of gene expression and loss-of-function (LOF) studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.

Coupling CRISPR interference with FACS enrichment: New approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production

“…FI of BFP was measured using microplate reader Infinite (Tecan), excited at 402 nm and emission measured at 464 nm. Clones with extremely high or low FI were excluded from further evaluation due to potential cytotoxic effect [ 35,36 ] or too low expression, respectively. Of the remaining clones, six clones with high FI and quantified dCas9 expression values (Figure S2) were selected for further transfections.…”

“…CHO-K1-derived proprietary parental cells were cultivated in proprietary, chemically defined serum-free medium in shake flasks and incubated at 37 or low FI were excluded from further evaluation due to potential cytotoxic effect [35,36] or too low expression, respectively. Of the remaining clones, six clones with high FI and quantified dCas9 expression values (Figure S2) were selected for further transfections.…”

Research and Therapeutic Approaches in Stem Cell Genome Editing by CRISPR Toolkit

“…This technology has been widely utilized to investigate the impact of changing gene expression levels on stem cell activity. In mouse ESCs, dCas9-Kruppel associated box (KRAB) inducible with doxycycline knock-in was used to study gene expression and repression, cell differentiation, or reprogramming [ 107 ]. In another investigation, in MSCs, CRISPR/dCas9-KRAB (CRISPRi) and CRISPR/dCas9- herpes simplex virus -based transcriptional activator VP64 domain (VP64) (CRISPRa) inducible with doxycycline (it has an inducible promoter that becomes activated with doxycycline) were used to control the expression of alkaline phosphatase (ALP) gene.…”

dCas9 techniques for transcriptional repression in mammalian cells: Progress, applications and challenges

“…[ 103 ] In addition, knocking down these core pluripotency transcription factors (SOX2 and NANOG) also caused cell differentiation which indicated by morphological changes and transient expression of mesoderm marker and neuronal progenitor marker. [ 41 ] CRISPRi‐mediated repression of the chromatin regulator MLL1 contributed to the reprogramming, [ 104 ] and the epigenetic age marker can be moved when somatic cells of human or mice are reprogrammed to iPSCs, which can be used to prevent or delay aging. [ 105 ]…”