title: Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).
abstract: Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.
authors.affiliation: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, VIC, Australia.
authors.forename: Caitlin A
authors.identifier: 0000-0002-0898-9904
authors.initials: CA
authors.lastname: Cooper
authors.affiliation: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, VIC, Australia.
authors.forename: Arjun
authors.initials: A
authors.lastname: Challagulla
authors.affiliation: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, VIC, Australia.
authors.forename: Kristie A
authors.initials: KA
authors.lastname: Jenkins
authors.affiliation: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, VIC, Australia.
authors.forename: Terry G
authors.initials: TG
authors.lastname: Wise
authors.affiliation: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, VIC, Australia.
authors.forename: Terri E
authors.initials: TE
authors.lastname: O'Neil
authors.affiliation: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, VIC, Australia.
authors.forename: Kirsten R
authors.initials: KR
authors.lastname: Morris
authors.affiliation: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, VIC, Australia.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Tizard
authors.affiliation: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, VIC, Australia. Timothy.Doran@csiro.au.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Doran
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002642:Chick Embryo; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005260:Female; D000072669:Gene Editing / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007315:Insemination, Artificial; D008297:Male; D000094704:RNA, Guide, CRISPR-Cas Systems; D013094:Spermatozoa / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: C110547:DMRT1 protein; D000094704:RNA, Guide, CRISPR-Cas Systems; D014157:Transcription Factors; D049452:Green Fluorescent Proteins
keywords: Avian models; CRISPR/Cas9; Gene editing; Genome engineering