Scripts used for paper [Evaluation of genome and base editing tools in maize protoplasts](https://www.frontiersin.org/articles/10.3389/fpls.2022.1010030/full), by Yannick Fierlej et al, *Front. Plant Sci.*, 2022.
This pipeline has been set up in order to detect the mutations caused by CRISPR and their frequencies from a collection of different sample of maize protoplasts. This pipeline is written in *Python* and can be used with *python 3.0*.
...
...
@@ -30,3 +32,27 @@ Some arguments can be passed to the pipeline. Some are needed and other are opti
```
CRISPR_mutalyser -f NP06_S6_R1_001.fastq -r NP06_S6_R2_001.fastq -t NP06_ZmKAK1_KAK1_233_L1750.fasta -a data/adaptors.fa -p primers_NP06.txt -pam 93 -d sens
```
## Paper abstract
The full text of the paper is available at [https://doi.org/10.3389/fpls.2022.1010030](https://doi.org/10.3389/fpls.2022.1010030).
**Introduction:** Despite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing
remains a labor-intensive process requiring often several months of *in vitro* culture to obtain mutant plantlets.
To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs
or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9)
prior to stable transformation, rapid analysis tools are helpful.
**Methods:** To this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools
coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established.
**Results and discussion:** Mutation types found with high frequency in maize leaf protoplasts had a trend
to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed
to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement,
that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing,
albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain
but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated
that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated
in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused
abnormal stomata, pale leaves and eventual plant death two months after sowing.
