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Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing.

ABSTRACT: In this report, we present an improved protocol for CRISPR/Cas9 genome editing in mice. The procedure consists in the electroporation of intact mouse zygotes with ribonucleoprotein complexes prepared in vitro from recombinant Cas9 nuclease and synthetic dual guide RNA. This simple cloning-free method proves to be extremely efficient for the generation of indels and small deletions by non-homologous end joining, and for the generation of specific point mutations by homology-directed repair. The procedure, which avoids DNA construction, in vitro transcription and oocyte microinjection, greatly simplifies genome editing in mice.

SUBMITTER: Teixeira M

PROVIDER: S-EPMC5764989 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing.

Teixeira Marie M Py Bénédicte F BF Bosc Christophe C Laubreton Daphné D Moutin Marie-Jo MJ Marvel Jacqueline J Flamant Frédéric F Markossian Suzy S

Scientific reports 20180111 1

In this report, we present an improved protocol for CRISPR/Cas9 genome editing in mice. The procedure consists in the electroporation of intact mouse zygotes with ribonucleoprotein complexes prepared in vitro from recombinant Cas9 nuclease and synthetic dual guide RNA. This simple cloning-free method proves to be extremely efficient for the generation of indels and small deletions by non-homologous end joining, and for the generation of specific point mutations by homology-directed repair. The p ...[more]

PMID: 29323173

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Project description:The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished via the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.

Project description:MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.

Dataset Information

Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing.

Publications

Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing.

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