Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9

Stajič, Ester (avtor), Kunej, Urban (avtor)

Genome editing techniques, such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9) are undoubtedly becoming an indispensable tool for improving food crops and tackling agricultural challenges. In the present study, key factors affecting transformation efficiency, such as PEG4000 concentration, incubation time, and plasmid amount were evaluated to achieve efficient delivery of CRISPR/Cas9 vector into cabbage protoplasts. Using amplicon sequencing, we confirmed a significant effect of PEG4000 concentration and incubation time on the induced target mutations. By optimizing the transformation protocol, editing efficiency of 26.4% was achieved with 40 µg of plasmid and 15 minutes incubation with 50% PEG4000. While these factors strongly affected the mutation rate, the viability of the transformed protoplasts remained high. Our findings would be useful for successful genome editing in cabbage and other brassicas, as well as in research areas such as gene function analysis and subcellular localization that rely on transient transformation methods in protoplasts.

• Jezik: Angleški jezik
• Ključne besede: CRISPR/Cas9, genome editing, protoplasts, PEG-mediated transformation, Brassica oleracea var. capitata
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: BF - Biotehniška fakulteta
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2023
• Št. strani: 9 str.
• Številčenje: Vol. 14, art. 1245433
• PID: 20.500.12556/RUL-151619
• UDK: 575
• ISSN pri članku: 1664-462X
• COBISS.SI-ID: 168169731
• Datum objave v RUL: 12.10.2023

Gradivo je del revije

• Naslov: Frontiers in plant science
• Skrajšan naslov: Front. plant sci.
• Založnik: Frontiers Research Foundation
• ISSN: 1664-462X
• COBISS.SI-ID: 3011663

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: zelje, urejanje genoma, genetika, protoplasti, genetske transformacije

Projekti

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: Z4-3215-2021
• Naslov: Optimizacija protokola za CRISPR/Cas9 in tarčno preurejanje nukleotidov pri zelju

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: P4-0077-2019
• Naslov: Kmetijske rastline - genetika in sodobne tehnologije