CRISPR/Cas9 genome editing introduction and optimization in the non-model insect Pyrrhocoris apterus

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60077344%3A_____%2F19%3A00506496" target="_blank" >RIV/60077344:_____/19:00506496 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/60076658:12310/19:43899510

Výsledek na webu

<a href="https://www.frontiersin.org/articles/10.3389/fphys.2019.00891/full" target="_blank" >https://www.frontiersin.org/articles/10.3389/fphys.2019.00891/full</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3389/fphys.2019.00891" target="_blank" >10.3389/fphys.2019.00891</a>

Jazyk výsledku

angličtina

Název v původním jazyce

CRISPR/Cas9 genome editing introduction and optimization in the non-model insect Pyrrhocoris apterus

Popis výsledku v původním jazyce

The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice Mus musculus, zebrafish Danio reiro, and the fruit fly Drosophila melanogaster. However, publications describing the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species Pyrrhocoris apterus. We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method, in vivo testing of sgRNA efficiency, and G0 germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.

Název v anglickém jazyce

CRISPR/Cas9 genome editing introduction and optimization in the non-model insect Pyrrhocoris apterus

Popis výsledku anglicky

The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice Mus musculus, zebrafish Danio reiro, and the fruit fly Drosophila melanogaster. However, publications describing the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species Pyrrhocoris apterus. We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method, in vivo testing of sgRNA efficiency, and G0 germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/GA15-23681S" target="_blank" >GA15-23681S: Neuroendokrinní regulace reprodukční diapauzy a metamorfózy hmyzu</a><br>

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Frontiers in physiology

ISSN

1664-042X

e-ISSN

—

Svazek periodika

10

Číslo periodika v rámci svazku

JUL 15

Stát vydavatele periodika

CH - Švýcarská konfederace

Počet stran výsledku

15

Strana od-do

891

Kód UT WoS článku

000475487600001

EID výsledku v databázi Scopus

2-s2.0-85069944729