AUTHOR=Angulo Juan , Astin Christopher P. , Bauer Olivia , Blash Kelan J. , Bowen Natalee M. , Chukwudinma Nneoma J. , DiNofrio Austin S. , Faletti Donald O. , Ghulam Alexa M. , Gusinde-Duffy Chloe M. , Horace Kamaria J. , Ingram Andrew M. , Isaack Kylie E. , Jeong Geon , Kiser Randolph J. , Kobylanski Jason S. , Long Madeline R. , Manning Grace A. , Morales Julie M. , Nguyen Kevin H. , Pham Robin T. , Phillips Monthip H. , Reel Tanner W. , Seo Jenny E. , Vo Hiep D. , Wukoson Alexander M. , Yeary Kathryn A. , Zheng Grace Y. , Lukowitz Wolfgang 

TITLE=CRISPR/Cas9 mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family

JOURNAL=Frontiers in Genome Editing

VOLUME=5

YEAR=2023

URL=https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2023.1251557

DOI=10.3389/fgeed.2023.1251557

ISSN=2673-3439

ABSTRACT=<p>Genome editing in plants typically relies on T-DNA plasmids that are mobilized by <italic>Agrobacterium</italic>-mediated transformation to deliver the CRISPR/Cas machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal settings, such as teaching labs. Gene-specific targeting sequences can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic or transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the <italic>Arabidopsis</italic> GROWTH-REGULATING FACTOR (GRF) genes, we were able to create a collection of predicted null mutations in all nine family members with little effort. We then explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. In our hands, multiplexing was associated with pronounced disparities: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species, thereby preventing a systematic mutagenesis of the family.</p>