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High fidelity CRISPR/Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells.

Abstract：

:Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chicken PGCs can be precise edited using donors containing CRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM). However, targeting was more efficient using SpCas9-HF1 when mutations were introduced only into the gRNA target sequence. We subsequently employed an eGFP-to-BFP conversion assay, to directly compare HDR mediated by SpCas9-WT and SpCas9-HF1 and discovered that SpCas9-HF1 increases HDR while reducing INDEL formation. Furthermore, SpCas9-HF1 increases the frequency of single allele editing in comparison to SpCas9-WT. We used SpCas9-HF1 to demonstrate the introduction of monoallelic and biallelic point mutations into the FGF20 gene and generate clonal populations of edited PGCs with defined homozygous and heterozygous genotypes. Our results demonstrate the use of oligonucleotide donors and high fidelity CRISPR/Cas9 variants to perform precise genome editing with high efficiency in PGCs.

journal_name

Sci Rep

journal_title

Scientific reports

authors

Idoko-Akoh A,Taylor L,Sang HM,McGrew MJ

doi

10.1038/s41598-018-33244-x

subject

Has Abstract

pub_date

2018-10-11 00:00:00

pages

15126

issue

1

issn

2045-2322

pii

10.1038/s41598-018-33244-x

journal_volume

8

pub_type

杂志文章

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