Abstract
Mouse parthenogenetic haploid embryonic stem cells (ESCs) are pluripotent cells generated from chemically activated oocytes. Haploid ESCs provide an opportunity to study the effect of genetic alterations because of their hemizygotic characteristics. However, their further application for the selection of unique phenotypes remains limited since ideal reporters to monitor biological processes such as cell differentiation are missing. Here, we report the application of CRISPR/Cas9-mediated knock-in of a reporter cassette, which does not disrupt endogenous target genes in mouse haploid ESCs. We first validated the system by inserting the P2A-Venus reporter cassette into the housekeeping gene locus. In addition to the conventional strategy using the Cas9 nuclease, we employed the Cas9 nickase and truncated sgRNAs to reduce off-target mutagenesis. These strategies induce targeted insertions with an efficiency that correlated with sgRNA guiding activity. We also engineered the neural marker gene Sox1 locus and verified the precise insertion of the P2A-Venus reporter cassette and its functionality by monitoring neural differentiation. Our data demonstrate the successful application of the CRISPR/Cas9-mediated knock-in system for establishing haploid knock-in ESC lines carrying gene specific reporters. Genetically modified haploid ESCs have potential for applications in forward genetic screening of developmental pathways.
Highlights
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has emerged as a simple and efficient genome editing method in vitro and in vivo[10,11,12,13]
We established mouse parthenogenetic haploid embryonic stem cells (ESCs), as described previously[3], since long-term culture of ESCs is associated with chromosomal abnormalities and epigenetic instability[27,28,29]
We examined the knock-in efficiency of the donor vector using these combinations of the Cas[9] types and single-guide RNA (sgRNA) in haploid ESCs (Table 1)
Summary
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has emerged as a simple and efficient genome editing method in vitro and in vivo[10,11,12,13]. The Cas[9] nuclease from Streptococcus pyogenes is directed to specific genomic loci through an engineered chimeric single-guide RNA (sgRNA) containing 20 nucleotides (nt) complementary to the target DNA sequence[10]. Several approaches based on the CRISPR/Cas[9] system have recently been developed to reduce undesired off-target mutagenesis; for example, the use of the Cas[9] nickase and truncated sgRNAs. The Cas9-D10A mutant nickase (Cas9n), in which aspartic acid at codon 10 is altered to alanine in the RuvCI nuclease domain, induces a nick in the genome, in contrast to DSB induction by Cas[910,19]. Double nicking by Cas9n with a pair of offset sgRNAs induces DSBs by reducing off-target mutagenesis[20,21,22,23] since individual nicks are preferably repaired by high fidelity base excision repair[24]. Our data demonstrate the feasibility of using recent CRISPR/Cas[9] techniques for engineering the haploid ES cell genome that could contribute to extending the range of future genetic screens
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