Mutagenesis of plant genome

Question 1

Principal of classic breeding

Answer 1

• chaning DNA is essential for breeding
• Traditional breeding: cross & select
  e.g: wild cabbage –> white cabbage, cauliflower, brussel sprouts, stem cabbage
  e.g: Teosinte –> corn
• Mutation breeding: Chamicals, radiation
  –> select progency that hast the wanted traits (characteristics)

Question 2

Difference between forward and reverse genetics

Answer 2

Foward genetics
Phenotype –> Genotype
* Starts with a mutant that has an interesting phenotype and then identifies the gene that is mutated
* mutation not knowing in the beginning
* random single-gene mutant

Reverse genetics
Genotype –> Phenotype
* make mutantion in known gene and then analyses what the effect of that mutation is on phenotype
* Phenotype not known in the beginning
* targeted mutagenesis to specific genes of interest

Question 3

Types of mutagens

Answer 3

Mutagen: a physical, chemical or biological agent capable of inducing changes in DNA called mutation.

physical = radiation, temp.
chemical = Metal ions, Alkylating agents (EMS)
bilogical = transposons, insertion sequences (IS), bacteria

Question 4

EMS mutagenesis

Answer 4

• EMS is a mutagen (most commonly chemical mutagen)
• produces a random mutation by changing G:C pairs to A:T pairs –> induced by guanine alkylation
• leads to point mutations
• induced throughout the genome
• EMS leads to 100 or 1000 of uncontrolled mutations in genome

Guanine Alkylation:
* Addition of Ethyl group onto the =O of guanine modifies it H-bonds
–> pairs with thymine instead of cytosine

Mutation:
* Mutations occure at tdifferent positions in the two alleles of chromomsome
* selfing is needed to obatin homozygous mutations

Example plant:
1. EMS to Seeds (M0)
2. Selfing of M1 (M1 x M1)
3. M2
4. Screening of Fruit of M2

Question 5

Mutations of DNA induced by radioactivity

Answer 5

Radioactivity can lead to:
* Single-strand break
* Base or sugar damage
* Double-strand break
* DNA-protein cross link
* DNA-DNA intra-strand break
* DNA-DNA inter-strand break
* large scale chromosomal alterations ( deletions, inversion..)

“atomic gardening” helped humankind to develop several highly wanted crops and vegetabels. (e.g Pasta noodle wheat)

Question 6

Principle of insertional mutagenesis and possible effects on genes
(e.g. knockdown, knockout)

Answer 6

Principal:
* creation of mutations by inserting one or more foreign baes pairs or longer DNA fragments into a gene
* diffrent types of DNAs can be inserted into a genome to induce mutations, depending on the organism
* Insertional mutations occurs naturally, by viruses or transposons, or can be artifically created for research purpose in the lab

Effects of inserting DNA into a gene:
* Enhanced expression –> knockup
* Reduced expression –> Knockdown

• Insertion in exon –> Knowckout (disturbed coding sequence expression)
• Insertion in Intron (oftenw ithout effect on gene expression)

Question 7

Transposon mutagenesis; difference between Retrotransposons and DNA transposons

Answer 7

Insertional mutagenesis in plants:
* Transposon mutagenesis
* T-DNA insertion mutagenesis

Transposon mutagenesis
* utilizes transposobale elements (TEs) that intergrate into a recipient genome to generate insertion mutations
* TEs: first identified by BArbara McClintock (NP: 1983) in Maize.
* TEs occure in almost all organisms

Transposable elements
* mobile gentic elemnts or jumong genes
* they are DNA sequences
* categorized in 2 classes, based on their transposition mechanism

2 Classes
Class 1: Retrotransposons mobilize via copy-and-paste manner through an RNA intermediate

Class 2: DNA-transposon usa a cut-and-paste mechanism

Retrotransposons increase their copy number more rapidly than DNA transposons.

Question 7

Retrotransposons

Answer 7

• RNA transpositio intermediate
• Reverse transcription created a DNA cops taht is randomly instered into a genome leading to muations
• Transposons amplify themsleves and therby can beckome abundant in genomes: Maize 49-78%
• Only present in eukrayotes

Question 8

DNA transposons

Answer 8

• used to introduce a piece of foreign DNA into a genome
• cut-and-paste mechanism

Question 9

Genome editing

Answer 9

Genome editing (GE):
* Modyfing genome at will
* remove or insert individuals nucleotides..
* remoe or insert shorter or longer stretches of DNA..
* mutate bases..
… at pre-defined postions in the genome

GE is based on the repair of a double-strand break
* Non-homologues end joining (NHEJ)
“self-healing capacity of the cell”
–> random changes, typically lead to gene inactivation

• Homology-dependent repair (HDR)
  with “repair DNA”
  –> precise gene editing: gene repair, gene inactivation or gene modification

*Double strand break occurs naturally at random positions –> NHEJ is a self healing capayity of the cell

Question 10

CRISPR-Cas9

Answer 10

CRISPR-CAS is a bacterial adaptive defence/immune system against viruses and plasmids. (Streptococcus pyogenes)

Nobelprize in Chemistry (2020) to Emmanuella Charpentier and Jennifer Doudna for “the development of ac method for genome editing”

CRISPR = Cluster Regulatory Interspaced Short Palindromic Repeats
CAS = CRISPR-associated Protein

What do we need:
* CAS 9 protein
* gRNA (guid RNA)
* PAM (protospacer adjacent motif) next to target sequence

not every sequence in the genome (pr a gene) can be targeted

Method:
1. Designe of gRNA
2. Delivery of CRISPR/Cas9 complex into target cell through e.g microinjection, electrophoration or viral vectors.
2. Cas9 cleavage at target site.
The gRNA guides thes Cas9 Enzyme to the specific target DNA sequence, recognition through PAM.
Cas9 induces DSB (Double strand break)
3. NHJE –> Cells natural repair machinery is engaged to repeair DSB. (knock-out)
or
HDR –> with donor DNA (knock-in)
4. Slection and screening
modiefied cells are selcted and screened for the successful intergration of the knock-in sequence.