Gene Cloning And Editing

Question 1

Web-based databases

Answer 1

Plantgbd
Ensembleplants
Gramene 
NCBI
Phytozome

Question 2

Benefits of web-based cloning

Answer 2

Allows for more specific and accurate experiments in tubes

Therefore less time consuming

Question 3

Ways to test for protein-protein interactions?

Answer 3

Yeast two hybrid system
Y2H library screening
Split ubiquitin system
Bimolecular Fluorescence Complementation (BiFC)

Question 4

Yeast 2-hybrid system

Answer 4

Produce fusion proteins with each of the yeast GAL4 protein domains. If the two proteins interact, the galactose metabolic genes will be activated. Reporter gene eg. LacZ should be used so that blue colour is expressed with the galactose metabolic enzyme. Additionally selectable marker such as HIS3/ADE2 allows growth in absence of histidine and adenine. Yeast strains for Y2H purposes now have LacZ and HIS3 artificially introduces.

Question 5

Y2H library screening

Answer 5

Is undertaken when second protein is unknown. So want to know which proteins interact with known protein’x’

Question 6

Pros and cons of Y2H systems?

Answer 6

Easy to find protein protein interaction. But everything needs to be directed to the yeast nucleus. Does not work with membrane spanning proteins. Also has large number of false positives and interactions should be confirmed with in vitro and in vivo assays.

Question 7

Split ubiquitin system

Answer 7

NubG attached to protein x and cub is attached to protiein y. If proteins interact, ubi will reassociate. When ubi ressoxiates PLV is cleaved and LexA printer is activated. The activation of lexA leads to ADE2 HIS3 and LacZ being expressed.

Question 8

Pros and cons of split ubiquitin system

Answer 8

Can be used to assay membrane proteins as interaction takes place in cytosine. Has less false positives than Y2H

Question 9

Bimolecular fluorescence complementation (BiFC)

Answer 9

GFP split into 2halves. Protein x is fused to the n terminus and protein y is fused to the c terminus. Co express both fusion proteins in plant cells. If there is an interaction between the proteins, fluorescence under blue light will be observed.

Question 10

Pros and cons of BiFC

Answer 10

Can see interaction in living cells. Transgenic lines don’t have to be produced( protoplasm tranfection, gene fun and agro infilitration can be undertaken).
Prone to false negatives as gfp halves have problems separating.

Question 11

Genome editing methods

Answer 11

ZFN, TALENS, CRISPR

Similarities: Targeted to specific DNA sequences. Use no -specific nucleases. Nucleases causes a single or double strand break, which triggers cellular DNA repair mechanisms( error prone non-homologous end joining and homology-directed repair)

Question 12

ZFN

Answer 12

Zinc fingers: protein domains that recognise a specific trinucleotode. ZFs designed for top and bottom sequences. Fokl fused to the cleavage domain.

Question 13

TALENS

Answer 13

34 as repeat modules. Each module contains 2 variable residues and each di-residue confers to a single nucleotide.

TALES are designed for top and bottom sequences and fokl is fused to cleavage domain.

Question 14

CRISPR/cas9

Answer 14

Cas9 nucleases joins RNA scaffold, CRISPR RNA recognises invaders DNA sequence and cas9 cuts DNA causing a double strand break. Double stranded breaks are repaired either by non-homologous end-joining or by homologous directed repair.

Cas9 requires a PAM sequence to bind to target DNA. Cas9 targets 20bases before PAM.

Question 15

PAM

Answer 15

DNA motif adjacent to DNA sequence targeted by cas9. Pam distinguishes self from non self and prevents CRISPR locus from being targeted.

Cas9 PAM is NGG on the 3’ side of the DNA sequence to be targeted.

Cpf1 PAM is TTN on the 5’ end of DNA sequence.

Question 16

Cas9 vs cpf1

Answer 16

Cas9 generates blunt ends 3nt upstream of PAM. SgRNA 100nt

Cpf1 generates 5nt 5’ overhangs 18-23 bases away from PAM. SgRNA 42nt

Question 17

CRISPR based systems

Answer 17

Base editing

Transcription control

Epigenome editing

Prime editing

Question 18

CRISPR BASE EDITING

Answer 18

Fuse dead cas9 to deaminase protein.

There is cytosine base editing (c/g goes to t/a) and adenine base editing (a/t goes to g/c). Deanination of adenine yields inosine which is treated as guanine by polymerases.

Many wild not gene knockouts, but single nucleotide polymorphisms (SNPs) examples: herbicide tolerance and nitrogen use efficiency.

Question 19

CRISPR Transcription control

Answer 19

Fuse dead cas9 with transcription activators and transcription receptors. Introduce mutations into promoter cis elements

Question 20

CRISPR epigenetic control

Answer 20

Dead cas9 fused with enzymes effecting epigentic status

DNA demethylase
Histone acetransferase
Dna methyltransferase
Histone demethylase

Question 21

Prime editing

Answer 21

Fuse nickase to a reverse transcriptase. Use a modified SgRNA that recognises target for Cas cutting and provides template for reverse transcriptase.

1. Nicking of Pam strand
2. Hybridisation of primer binding site to PAM strand
3. Reverse transcriptase
4. Hybridisation of DNA strands and flap cleavage
5. Ligation and mismatch repair

Allows for substitutions, deletions, insertions and completely new sequences.

Question 22

CRISPRing plants

Answer 22

Need to express both CRISPR elements in plants and produce transgenic plants.

Agrobacterum mediated transformation.
Antibiotic selection
Muattaionbdetect in t1 line. Single copy of t DNA insert is ideal.
Screen for homozygous in t2 progeny

Question 23

CRISPR pros and cons

Answer 23

Efficient at non homologous end joining. But doesn’t work for homologous directed repair.

Not too many off targets

Main challenge is the delivery method as GM generation must be produces and many crops are difficult to transform.

Government regulations mean that non GM is often required. And non-go mediated CRISPR is of interest to researchers