How to use CRISPR/Cas9 in plants: from target site selection to DNA repair.

A tool for precise, target-specific, efficient, and affordable genome editing is a dream for many researchers, from those who conduct basic research to those who use it for applied research. Since 2012, we have tool that almost fulfils such requirements; it is based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight key factors affecting its mutagenesis outcomes: (i) factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state, or Cas9 variant, and how long it remains in place after cleavage; and (ii) factors affecting the follow-up DNA repair mechanisms including mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts, and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.

DNA methylation can alter CRISPR/Cas9 editing frequency and DNA repair outcome in a target-specific manner.

The impact of epigenetic modifications on the efficacy of CRISPR/Cas9-mediated double-stranded DNA breaks and subsequent DNA repair is poorly understood, especially in plants. In this study, we investigated the effect of the level of cytosine methylation on the outcome of CRISPR/Cas9-induced mutations at multiple Cas9 target sites in Nicotiana benthamiana leaf cells using next-generation sequencing. We found that high levels of promoter methylation, but not gene-body methylation, decreased the frequency of Cas9-mediated mutations. DNA methylation also influenced the ratio of insertions and deletions and potentially the type of Cas9 cleavage in a target-specific manner. In addition, we detected an over-representation of deletion events governed by a single 5'-terminal nucleotide at Cas9-induced DNA breaks. Our findings suggest that DNA methylation can indirectly impair Cas9 activity and subsequent DNA repair, probably through changes in the local chromatin structure. In addition to the well described Cas9-induced blunt-end double-stranded DNA breaks, we provide evidence for Cas9-mediated staggered DNA cuts in plant cells. Both types of cut may direct microhomology-mediated DNA repair by a novel, as yet undescribed, mechanism.

Unexpected variations in posttranscriptional gene silencing induced by differentially produced dsRNAs in tobacco cells.

In plants, posttranscriptional gene silencing (PTGS) is induced by small RNAs (sRNAs) generated from various dsRNA precursors. To assess the impact of dsRNA origin, we compared downregulation of GFP expression triggered by inverted repeat (IR), antisense (AS) and unterminated sense (UT) transcripts transiently expressed from the estradiol-inducible promoter. The use of homogeneously responding tobacco BY-2 cell lines allowed monitoring the onset of silencing and its reversibility. In this system, IR induced the strongest and fastest silencing accompanied by dense DNA methylation. At low induction, silencing in individual cells was binary (either strong or missing), suggesting that a certain threshold sRNA level had to be exceeded. The AS variant specifically showed a deviated sRNA-strand ratio shifted in favor of antisense orientation. In AS lines and weakly induced IR lines, only the silencer DNA was methylated, but the same target GFP sequence was not, showing that DNA methylation accompanying PTGS was influenced both by the level and origin of sRNAs, and possibly also by the epigenetic state of the locus. UT silencing appeared to be the least effective and resembled classical sense PTGS. The best responding UT lines behaved relatively heterogeneously possibly due to complexly arranged T-DNA insertions. Unlike IR and AS variants that fully restored GFP expression upon removal of the inducer, only partial reactivation was observed in some UT lines. Our results pointed out several not yet described phenomena and differences between the long-known silencer variants that may direct further research and affect selection of proper silencer variants for specific applications.

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells.

BACKGROUND: Methylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert. RESULTS: We show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases. CONCLUSIONS: Our results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.