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. 2016 Feb;14(2):519-32.
doi: 10.1111/pbi.12468. Epub 2015 Sep 11.

Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis

Yanfei Mao  1 , Zhengjing Zhang  1   2 , Zhengyan Feng  1   2   3 , Pengliang Wei  1   2 , Hui Zhang  1 , José Ramón Botella  4 , Jian-Kang Zhu  1   5
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Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis

Yanfei Mao et al. Plant Biotechnol J. .
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Abstract

The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.

Keywords: Arabidopsis; CRISPR-Cas9; gene modification; germ-line-specific.

Figures

Figure 1
Schematic representation of the workflow designed to detect targeted gene mutations in the Arabidopsis T1 population. For each of the eight CRISPR binary constructs, 32 individual Arabidopsis transgenic T1 lines were subjected to mutation detection by Sanger sequencing of PCR products amplified with primers flanking the sgRNA target sites from both leaf and floral tissue samples. For T1 chimeras, analysis was performed by examining their sequencing chromatograms. A typical chromatogram for a T1 chimera shows single peaks extending up to the sgRNA target site but immediately after the target site the quality of the chromatogram dramatically decreases and multiple peaks start to appear in each nucleotide position. HQ%: proportion of high-quality reads. The arrow head indicates the position of sgRNA target site.
Figure 2
Gene mutagenesis efficiency of the GSC and UC CRISPR/Cas9 systems in T1 and T2 generations. (a), (b) Frequency of targeted gene mutations at the AP1 (a) and TT4 (b) loci in plants transformed with the two CRISPR/Cas9 systems. Thirty-two individual T1 lines were analysed for each transformation. DNA was extracted from leaf and floral tissues separately. For the T1 generation, 32 plants were analysed and mutation frequencies calculated for each locus. For the T2 generation, eight individuals from each T1 line were pooled for mutation detection by sequencing. (c), (d) Sequencing chromatograms of T1 chimeras generated by the UC system (upper line) and the GSC system (lower line) showing mutations at the AP1-24 (c) or the TT4-296 (d) site. The HQ % value of each chromatogram is on the right side. HQ%: proportion of high-quality reads. The arrows indicate the position of sgRNA target sites.
Figure 3
Genotyping of targeted gene mutations induced by the GSC and UC CRISPR/Cas9 systems in the T2 generation. (a) Mutagenesis frequencies for all four targeted loci. The mutagenesis frequency was calculated as the percentage of mutated plants in T2 progenies derived from eight individual T1 lines. (b) Combined data for all four loci showing mutagenesis frequency of both CRISPR/Cas9 systems and the proportion of chimeras present in the mutant population. (c) Zygosity of targeted gene mutations in T2 populations. The frequencies of each zygotic type are shown as percentages of the overall T2 mutant population. (d) Average HQ% value of the chromatograms for T2 chimera generated by the UC and GSC systems grouped by target gene. HQ% represents the proportion of high-quality reads. Error bar = SE.
Figure 4
Gene mutagenesis efficiency of the modified CRISPR/Cas9 systems in T1 and T2 generations. (a) Frequency of targeted gene mutations at the AP1-24 and TT4-296 loci in plants transformed with the either the IUC system or the original UC system. (b) Comparative analysis of the GEC and the original GSC systems. (c), (d) Sequencing chromatograms of T1 chimeras generated by the GEC system showing the highest HQ% value (upper line) and the lowest HQ% value (lower line) at the AP1-24 locus (c) and the TT4-296 locus (d). Line numbers are shown on the left of the chromatograms and the HQ % values on the right side. The arrows indicate the position of sgRNA target sites.
Figure 5
Genotyping for targeted gene mutations induced by the modified CRISPR/Cas9 systems in the T2 generation. (a) Mutagenesis frequencies for all four targeted loci in T2 progenies generated by the UC and IUC systems. The mutagenesis frequency was calculated as the percentage of mutated plants in T2 progenies derived from 8 individual T1 lines. (b) Similar analysis performed with T2 plants generated by the GSC and GEC systems. (c) Zygosity of targeted gene mutations in T2 populations generated by the UC and IUC systems. The frequencies of each zygotic type are shown as percentages of the overall T2 mutant population. (d) Zygosity of targeted gene mutations in T2 populations generated by the GSC and GEC systems. The frequencies of each zygotic type are shown as percentages of the overall T2 mutant population.
Figure 6
Gene mutagenesis efficiency of the gamete-specific DD45 and LAT52 CRISPR/Cas9 systems in T1 and T2 generations. (a) Frequency of targeted gene mutations at the GL2-89 and GL2-97 loci in T1 plants using the DD45 and LAT52 systems. Mutants of different zygotic types are shown in different colour. (b) Sequencing chromatograms of the three heritable T1 mutants generated by the DD45 system. The zygotic types and mutant line numbers are shown on the left side. The arrows indicate the position of sgRNA target sites. (c) Mutation frequency in 24 T2 individuals generated by the DD45 and LAT52 systems. (d) Zygosity of T2 progeny.
Figure 7
A model showing that the GSC system is capable of generating more diverse mutations compared to the UC system. In the ubiquitous CRISPR/Cas9 system, the T1 mutants are usually chimeras combining multiple mutation types. As most of the mutations happen in somatic cells, only a small percentage of the mutation types that occur in the progenitor cells of the gametocytes can be inherited by their progenies. Thus, the diversity of the targeted gene mutations is limited. In contrast, in the SPL germ-line-specific system, T1 mutations are induced mainly in microsporocytes, so that the NHEJ-based gene repair process is independently induced in each microspore expressing CRISPR/Cas9. Considering the large quantity of pollen cells, application of this system will strongly increase the diversity of heritable gene mutations in the T2 population.
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