Abstract
CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using expression vectors, mRNA, or directly as ribonucleoprotein (RNP) complexes by different delivery methods. Whereas microinjection techniques are well established, more recently developed electroporation methods simplify RNP delivery but can provide less consistent efficiency. Previously, we have designed Cas12a-crRNA pairs to introduce large genomic deletions in the Ubn1, Ubn2, and Rbm12 genes in mouse embryonic stem cells (ESC). Here, we have optimized the conditions for electroporation of the same Cas12a RNP pairs into mouse zygotes. Using our protocol, large genomic deletions can be generated efficiently by electroporation of zygotes with or without an intact zona pellucida. Electroporation of as few as ten zygotes is sufficient to obtain a gene deletion in mice suggesting potential applicability of this method for species with limited availability of zygotes.
Highlights
Clustered regularly interspaced short palindromic repeat (CRISPR) associated nucleases are components of bacterial and archaeal adaptive defense mechanisms against invasive nucleic acids such as plasmids or viral DNA (Horvath and Barrangou 2010)
The NEPA electroporation system has been previously used for zygote electroporation (Hashimoto and Takemoto 2015; Hashimoto et al 2016; Hur et al 2016) and is available with electroporation chambers that are designed for use with embryos
In our study we developed a method for small volume zygote electroporation of Cas12a-CRISPR RNAs (crRNA) RNPs and demonstrate its use for generating large gene deletions
Summary
Clustered regularly interspaced short palindromic repeat (CRISPR) associated nucleases are components of bacterial and archaeal adaptive defense mechanisms against invasive nucleic acids such as plasmids or viral DNA (Horvath and Barrangou 2010) These nuclease enzymes associate with short CRISPR RNAs (crRNA) that guide them to complementary target sequences in the genome. As the CRISPR associated nucleases Cas and Cas12a can be programmed with crRNA sequences of choice, they have been repurposed into a versatile genome editing tool for many biological tissues in different species (Hai et al 2014; Hwang et al 2013; Jinek et al 2012; Lee et al 2019; Mali et al 2013; Sung et al 2014; Wang et al 2013). These studies suggest that ESC technology might be replaced by direct engineering of the zygotic genome for gene targeting
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
