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TIE: A Method to Electroporate Long DNA Templates into Preimplantation Embryos for CRISPR-Cas9 Gene Editing.

ABSTRACT: Precise genome editing using CRISPR typically requires delivery of guide RNAs, Cas9 endonuclease, and DNA repair templates. Both microinjection and electroporation effectively deliver these components into mouse zygotes provided the DNA template is an oligonucleotide of only a few hundred base pairs. However, electroporation completely fails with longer double-stranded DNAs leaving microinjection as the only delivery option. Here, we overcome this limitation by first injecting all CRISPR components, including long plasmid-sized DNA templates, into the sub-zona pellucida space. There they are retained, supporting subsequent electroporation. We show that this simple and well-tolerated method achieves intracellular reagent concentrations sufficient to effect precise gene edits.

SUBMITTER: Bagheri H

PROVIDER: S-EPMC6636866 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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TIE: A Method to Electroporate Long DNA Templates into Preimplantation Embryos for CRISPR-Cas9 Gene Editing.

Bagheri Hooman H Friedman Hana H Shao Harry H Chong Yumaine Y Lo Chiu-An CA Emran Farida F Kays Ibrahim I Yang Xiang-Jiao XJ Cooper Ellis E Chen Brian E BE Siminovitch Katherine K Peterson Alan A

The CRISPR journal 20180601

Precise genome editing using CRISPR typically requires delivery of guide RNAs, Cas9 endonuclease, and DNA repair templates. Both microinjection and electroporation effectively deliver these components into mouse zygotes provided the DNA template is an oligonucleotide of only a few hundred base pairs. However, electroporation completely fails with longer double-stranded DNAs leaving microinjection as the only delivery option. Here, we overcome this limitation by first injecting all CRISPR compone ...[more]

PMID: 31021258

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