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CRISPR_mutalyser
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README.md

Mutation analysis

Scripts used for paper Evaluation of genome and base editing tools in maize protoplasts, 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.

Program version

This pipeline uses several external tools, here is the version numbers of the used software:

Syntax

Some arguments can be passed to the pipeline. Some are needed and other are optional:

  • -h or --help : show help message and exit
  • -f or --forward (required): forward file of the reads for the assembly
  • -r or --reverse (required): reverse file of the reads for the assembly
  • -a or --adapters (required): fasta sequence of the adapters
  • -t or --target (required): fasta sequence of the target sequence for the alignment
  • -p or --primer (required): fasta sequence of the primer (as to be the same number of file as the sequence target)
  • -pam (required): position of the first base of the PAM sequence (used to know the crispr range)
  • -o or --directory (optional): path to the output results (default : /output_program/)
  • -cutoff (optional)(0-1): cutoff for the frequency logo (default: 0.0001)
  • -score (optional)(0-350): minimal alignment score required for the analyse (default: 200), can't be higher than 350
  • -d or --direction (required) (sens,antisens,forward,reverse): direction of the PAM sequence (as to be the same number of file as the number of PAM positions)

Command line

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.

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.