Exploiting the CRISPR/Cas9 System for Targeted Genome Mutagenesis in Petunia

Abstract

Recently, CRISPR/Cas9 technology has emerged as a powerful approach for targeted genome modification in eukaryotic organisms from yeast to human cell lines. Its successful application in several plant species promises enormous potential for basic and applied plant research. However, extensive studies are still needed to assess this system in other important plant species, to broaden its fields of application and to improve methods. Here we showed that the CRISPR/Cas9 system is efficient in petunia (Petunia hybrid), an important ornamental plant and a model for comparative research. When PDS was used as target gene, transgenic shoot lines with albino phenotype accounted for 55.6%-87.5% of the total regenerated T0 Basta-resistant lines. A homozygous deletion close to 1 kb in length can be readily generated and identified in the first generation. A sequential transformation strategy-introducing Cas9 and sgRNA expression cassettes sequentially into petunia-can be used to make targeted mutations with short indels or chromosomal fragment deletions. Our results present a new plant species amenable to CRIPR/Cas9 technology and provide an alternative procedure for its exploitation.During the past three decades, great efforts and achievements have been made in developing efficient tools for targeted genome modification in plants 1,2 . Before the CRISPR/ Cas9 system (CRISPR: clustered regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9) was introduced into plant research in 2013 3-10 , three classes of sequence-specific nucleases had been used for plant genome engineering: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and meganucleases 11 . Against this background, due to its versatility, design simplicity, high efficiency and low cost, the CRISPR/Cas9 technique has rapidly become a focus of attention over the past three years 1,2,[12][13][14][15] . The CRISPR/Cas9 system is composed of Cas9 nuclease and customizable sgRNA. The sgRNA guides Cas9 to recognize target DNA and create double strand breaks (DSBs) which trigger non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways, resulting in genome modifications 16 . Genome modification is based on the repair of DSBs 11,17 . Because previous reports show that the efficiency of DSB repair pathways differs between species and also cell types 1,17 , it is important to investigate the feasibility of the CRISPR/ Cas9 system in each plant species of interest.To date, the primary application of the CRISPR/Cas9 system in plants has been the creation of gene knock-outs 13,15 . It has been successfully used to induce genetic modification in plants including Arabidopsis 3,[6][7][8][18][19][20] 34 . These reports show that CRISPR/ Cas9-mediated targeted mutagenesis is efficient in plants. Analysis also suggests that the production of mutations by CRISPR/Cas9 in plants is highly specific 20,23 and can be stably transmitted to subsequent generations following classic Mendelian law [18][19][...

Patterns of CRISPR/Cas9 activity in plants, animals and microbes

“…The reported efficiency of indels induced by CRISPR/Cas9 in both dicots and monocots can vary significantly even within the same species (e.g. 1.1%–90.4% in Arabidopsis), but most species offer examples of efficiency approaching 100%, including Arabidopsis (Yan et al ., ), soya bean (Cai et al ., ), potato (Wang et al ., ), tomato (Pan et al ., ), petunia (Zhang et al ., ), tobacco (Gao et al ., ), poplar (Zhou et al ., ), grapefruit (Jia et al ., ), maize (Svitashev et al ., ) and rice (Ma et al ., ). Notably, the CRISPR/Cas9 system can efficiently induce mutations in different tissues and cell types, including embryogenic callus (e.g.…”

“…But the frequency of heritable mutations increased to 90.4% when the constitutive promoter was replaced with an egg‐cell‐specific promoter (Wang et al ., ), a cell‐division‐specific promoter (Hyun et al ., ; Yan et al ., ) or a germ‐line‐specific promoter (Mao et al ., ). In all other plant systems, constitutive promoters have achieved high mutation frequencies, with biallelic and homozygous mutants readily produced in the first generation of monocots such as maize (Svitashev et al ., ) and rice (Lowder et al ., ; Ma et al ., ; Miao et al ., ; Wang et al ., ) and dicots such as tomato (Brooks et al ., ; Pan et al ., ), poplar (Fan et al ., ; Zhou et al ., ), potato (Wang et al ., ), petunia (Zhang et al ., ) and tobacco (Gao et al ., ). These observations also suggest that mutagenesis often occurs at an early stage during the transformation process, before the first cell division.…”

Applications and roles of the CRISPR system in genome editing of plants

“…Auxin response factor 1 (MpARF1) (Sugano et al 2014) Petunia Petunia hybrid Phytoene desaturase (PDS) (Zhang et al 2016a) 2013, 2015). CRISPR/Cas9 has proven to be extremely versatile because only a short synthetic RNA sequence is needed to recognize a new target (Belhaj et al 2013(Belhaj et al , 2015Bortesi and Fischer 2015;Liang et al 2014;Wang et al 2015).…”

“…Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100 %) . Zhang et al (2016a, b) studied the efficiency of the TALEN system in rice and the nature and inheritability of TALENinduced mutations and described important features of this technology. The N287C230 TALEN backbone resulted in low mutation rates (0-6.6 %), but truncations in its C-terminal domain greatly increased the efficiency to 25 %.…”

Naming CRISPR alleles: endonuclease-mediated mutation nomenclature across species

“…(2013), Straimer et al (2012), reviewed in Singer and Frischknecht (2017) RoundwormCRISPR, TALEN, ZFNreviewed in Gaj et al (2013), Waaijers and Boxem (2014) Sea squirtCRISPR, TALEN, ZFNKawai et al (2012), Sasaki et al (2014), Treen et al (2014) SilkwormCRISPR, TALEN, ZFNReviewed in Xu and O’Brochta (2015)Plants BarleyCRISPR, TALENLawrenson et al (2015), Wendt et al (2013) BunchgrassTALENShang et al (2013a) CabbageCRISPR, TALENLawrenson et al (2015), Sun et al (2013) CornCRISPR, TALEN, ZFNChar et al (2015), Shukla et al (2009), Somaratne et al (2017) ChlamydomonasCRISPR, ZFNShin et al (2016), Sizova et al (2013) Duncan grapefruitCRISPRJia et al (2016) LiverwortCRISPRSugano et al (2014) PetuniaCRISPR, ZFNMarton et al (2010), Zhang B et al (2016a, b, c) RiceCRISPR, TALENLi et al (2012), reviewed in Belhaj et al (2013) SorghumCRISPRJiang et al (2013) SoybeanCRISPR, TALEN, ZFNCurtin et al (2011), Haun et al (…”