Repositorium

What is a repositorium?

The repositorium is a searchable database that provides data on relevant articles from journals, company web pages and web pages of governmental agencies about studies/applications of genome-editing in model plants and agricultural crops in the period January 1996 to May 2018. Search options are article type, technique, plant, traits or free text. The repositorium is based on the systematic map of Dominik Modrzejewski et al., published in the journal environmental evidence. (Download article PDF).

High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca sativa)


Typ / Jahr

Journal Article / 2018

Autoren

Bertier, Lien D.; Ron, Mily; Huo, Heqiang; Bradford, Kent J.; Britt, Anne B.; Michelmore, Richard W.

Abstract
CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4), a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4, were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T1) and 368 T2 plants by deep amplicon sequencing revealed that 57% of T1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T1 and more than 100 T2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature.
Keywords
CRISPR/Cas9; NHJE; targeted mutagenesis; lettuce; germination; thermotolerance; amplicon sequencing
Periodical
G3 (Bethesda, Md.)
Periodical Number
5
Page range
1513–1521
Volume
8
DOI
10.1534/g3.117.300396

Techniques

ID Corresponding Author
Country
Plant Species GE Technique
Sequence Identifier
Trait
Type of Alteration
Progress in Research
Key Topic
670 Michaelmore, Richard W.
USA
Lactuca sativa CRISPR/Cas9
NCED4
Germination at high temperature
SDN1
Market-oriented
Agronomic value