Efficient mouse genome engineering by CRISPR-EZ technology

Modzelewski, A; Chen, Sean; Willis, Brandon; Lloyd, Kevin C K; Wood, Joshua A.; He, Lin

doi:10.1038/nprot.2018.012

Cited by 101 publications

(94 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Compared to the previously described microinjection-based technique, several zygotes (30 to 50) can be simultaneously genome edited using a square pulse generator (electroporator), a technique that does not require expensive micromanipulator systems. Detailed protocols for EP-based genome editing have been reported by Kaneko [43], Qin et al [44], and Modzelewski et al [45].…”

Section: Ep Techniquementioning

confidence: 99%

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

Takabayashi

Akasaka

Nakamura

2020

Cells

View full text Add to dashboard Cite

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of efficient and simple methods to produce genome-edited (GE) animals would accelerate research in this field. The CRISPR/Cas9 system was initially employed in early embryos, utilizing classical gene delivery methods such as microinjection or electroporation, which required ex vivo handling of zygotes before transfer to recipients. Recently, novel in vivo methods such as genome editing via oviductal nucleic acid delivery (GONAD), improved GONAD (i-GONAD), or transplacental gene delivery for acquiring genome-edited fetuses (TPGD-GEF), which facilitate easy embryo manipulation, have been established. Studies utilizing these techniques employed pregnant female mice for direct introduction of the genome-editing components into the oviduct or were dependent on delivery via tail-vein injection. In mice, embryogenesis occurs within the oviducts and the uterus, which often hampers the genetic manipulation of embryos, especially those at early postimplantation stages (days 6 to 8), owing to a thick surrounding layer of tissue called decidua. In this review, we have surveyed the recent achievements in the production of GE mice and have outlined the advantages and disadvantages of the process. We have also referred to the past achievements in gene delivery to early postimplantation stage embryos and germ cells such as primordial germ cells and spermatogonial stem cells, which will benefit relevant research.

show abstract

Section: Ep Techniquementioning

confidence: 99%

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

Takabayashi

Akasaka

Nakamura

2020

Cells

View full text Add to dashboard Cite

show abstract

“…A homology directed repair (HDR) donor DNA oligo with the desired mutation was synthesized by Life Technologies Co., with a single T!C point mutation that converts a tryptophan (TGG) to arginine (CGG) at position 97 in the L d gene. Cas9-sgRNA RNP was prepared and incorporated together with the HDR donor DNA oligo and electroporated into mouse zygotes from H-2 d (B6.C) mice as described (Chen et al 2016;Modzelewski et al 2018). Resulting mice were genotyped by sequencing.…”

Section: Generation Of L D W97r Mutant Mouse By Crispr/cas9mentioning

confidence: 99%

“…Generation of L d W97R mutant mouse by CRISPR-EZ (Chen et al 2016;Modzelewski et al 2018). Cas9-sgRNA ribonucleoprotein (RNP) was prepared and incorporated together with the HDR donor DNA oligo and electroporated into mouse zygotes from H-2 d (B6.C) mice.…”

Section: Figure 4: Polymorphism In H2-l D Increases Self-peptide Bindmentioning

confidence: 99%

An unusual MHC molecule generates protective CD8+ T cell responses to chronic infection

Tsitsiklis

Bangs

Lutes

et al. 2020

Preprint

View full text Add to dashboard Cite

The CD8+ T cell response to the intracellular parasite Toxoplasma gondii varies dramatically between mouse strains, resulting in differences in control of the parasite. Protection in BALB/c mice can be attributed to an unusually strong and protective MHC-1 L d -restricted CD8+ T cell response directed against a peptide derived from the parasite antigen GRA6. The MHC-1 L d molecule has limited peptide binding compared to conventional MHC molecules such as K b or D b , which correlates with polymorphisms associated with "elite control" of HIV in humans. To investigate the link between the unusual MHC-1 molecule L d and the generation of "elite controller" CD8+ T cell responses, we compared the GRA6-L d specific T cell response to the well-studied OVA-K b specific response, and demonstrated that GRA6-L d specific T cells are significantly more protective and resistant to exhaustion in chronic T. gondii infection. To further investigate the connection between limited peptide presentation and robust T cell responses, we used CRISPR/Cas9 to generate mice with a point mutation (W97R) in the peptide-binding groove of L d that results in broader peptide binding. We investigated the effect of this L d W97R mutation on another robust L d -restricted response against the IE1 peptide during Murine Cytomegalovirus (MCMV) infection. This mutation leads to an increase in exhaustion markers in the IE1-L d specific CD8+ T cell response. Our results indicate that limited peptide binding by MHC-1 L d correlates with the development of robust and protective CD8+ T cell responses that may avoid exhaustion during chronic infection.

show abstract

“…The CRISPR-EZ method, where single guide RNA (sgRNA)/Cas9 complexes are delivered into mouse zygotes by electroporation, was developed by Chen et al and Modzelewski et al 11,12 .…”

Section: Generation Of Abhd2 Ko Mice Using Crispr Ribonucleoprotein Ementioning

confidence: 99%

“…To study this further, we have generated an ABHD2 knockout mouse by using the highly efficient CRISPR Ribonucleoprotein Electroporation of Zygotes (CRISPR-EZ) technique 11,12 and evaluated its fertility phenotype. We show that ABHD2 is needed to regulate the cyclic maturation of follicles.…”

Section: Introductionmentioning

confidence: 99%

Alpha/Beta Hydrolase Domain-Containing Protein 2 regulates the rhythm of follicular maturation and estrous stages of the female reproductive cycle

Björkgren

Chung

Mendoza

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Therian female fertility is defined by a successful and strictly periodic ovarian cycle, which is under the control of gonadotropins and steroid hormones, particularly progesterone and estrogen. The latter two are produced by the ovaries that are engaged in controlled follicular growth, maturation and release of the eggs, i.e. ovulation. It is well known that steroid hormones regulate ovarian cycles via genomic signaling, by altering gene transcription and protein synthesis. However, despite this well-studied mechanism, steroid hormones can also signal via direct, non-genomic action, by binding to their membrane receptors. Here we show, that the recently discovered sperm membrane progesterone receptor α/β hydrolase domain-containing protein 2 (ABHD2) is highly expressed in mammalian ovaries where the protein plays a novel regulatory role in follicle maturation and the sexual cycle of females. Ablation of Abhd2 caused a dysregulation of the estrous cycle rhythm with females showing shortened luteal stages while remaining in the estrus stage for a longer time. Interestingly, the ovaries of Abhd2 knockout (KO) females resemble polycystic ovary morphology with a high number of atretic antral follicles that could be rescued with injection of gonadotropins. Such a procedure also allowed Abhd2 KO females to ovulate a significantly increased number of mature and fertile eggs in comparison to their wild-type littermates. These results suggest a novel regulatory role of ABHD2 as an important factor in nongenomic steroid regulation of the female reproductive cycle. Results Abhd2 expression in ovarian stromal cells and corpora luteaWhile ABHD2 has been described as an important regulator of sperm function 10 , the function of ABHD2 in female reproduction was not known. Interestingly, unlike the male reproductive tissues, we found that ABHD2 is not detected in the female gametes but is predominantly expressed in the stromal cells surrounding the developing follicles (Fig. 1a). The presence of both ABHD2 protein and mRNA was observed in pre-pubertal mouse ovaries, as shown here by immunohistochemical (IHC) staining and reverse transcriptase quantitative PCR (RT-qPCR; Fig. 1b and c). Around one month after birth, when the ovulatory cycle begins, ABHD2 expression is further found in the lutein cells of corpus luteum (Fig. 1a). In correlation with this, qPCR studies showed highest Abhd2 mRNA presence right after ovulation during the estrus stage (Fig. 1c), and the expression levels are even further increased after induction of superovulation by gonadotropin injection (Fig. 1c).

show abstract

Efficient mouse genome engineering by CRISPR-EZ technology

Cited by 101 publications

References 54 publications

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

An unusual MHC molecule generates protective CD8+ T cell responses to chronic infection

Alpha/Beta Hydrolase Domain-Containing Protein 2 regulates the rhythm of follicular maturation and estrous stages of the female reproductive cycle

Contact Info

Product

Resources

About