U4: Gene cloning & Editing

Cloning, CRISPR, GMO, Editing

What are the uses of gene cloning?

creating purified proteins, investigating mutations, manipulating DNA for insertion into organism, making DNA or cDNA libraries for genome/ RNA sequencing.

Parts of Plasmid

Promoter, Recognition site, recombination site, Antibiotic resistance, Replication origin. Repressor sequences (some plasmids only)

Vectors

Plasmids that contain own genes that are not apart of chromosome. Replicate on own and useful for inserting genes into cells. 

Antibiotic resistance

Selectable “marker” that selects for transgenic cells. Bacteria will survive use used antibiotics.

Replication Origin

Needed for plasmid to replicate and continue growing

Promoter

The transcription initiation of plasmids

Recombinant DNA

DNA from two different sources combined as one

DNA Palindrome

Both the forward and backward complementary DNA are the same sequences.

Sticky end DNA

Single stranded DNA overhang that attaches to base pair complements to each other (e.g. GAATTC). Restriction enzyme —> sticky ends —> ligase combines

Cloning Steps

Isolate plasmid (via enzyme) —> Cut plasmid (same enzyme) —> Ligate together —> insert into bacteria + antibiotic gene.

How to ensure your gene has restriction sites?

PCR primers allow for inserting restriction sites. 3’ end primer needs to be complementary to target sequence & 5’ end include any sequence/ new restriction sites. Sites are used to digest then ligate together

Pros of using different restriction enzymes for different sites?

Correct orientation of how the gene is inserted (sticky ends are cut by different enzymes can fit only one way), Has the gene of interest included into the plasmid.

Cons of using same enzymes for restriction sites?

The sticky ends can just recirculate (combining together) causing no recombination.

Gibson Assembly

Restriction site/enzyme NOT needed; PCR first —> Exonuclease removed 5’ end —> anneal —> DNA would base pair with each other.

Pros of Gibson Assembly?

Beneficial when trying to combine multiple fragments (doesn’t need specific site of each gene) PCR match up ends of the same sequences and make it one continues piece of DNA

Gene Editing

Complete alternation of genes that are usually passed down into offsprings

CRISPR Enzymes

Cut DNA based on guide RNAs. Body natural repones to target specific virial DNA, the CRISPR locus keep track of viral DNA.

Gene Editing w/Cas9

Cas9 causes a double strand break using guide RNA (very specific target of sequence in genome)

Non-homology end joining (NHEJ)

Insertion/deletion gene disruption (more common)

Homology-directed repair (HDR)

Precise DNA editing gene insertion

How to increase efficiency of HDR?

nicking Cs9 enzyme causes it to function as single strand break instead of DSB and forcing a homologous recombination.

Modified Cas9 Pros?

Cas9 with a defective nuclease can act as sequence detectors for other enzymes by removing its ability to cut.

Prime Editing

Uses reverse transcriptase and Cas9 by nicking DNA and adding in new edited sequence made by RT. FEW BASES not for long sequence editing.

Gene Drive

Genetically encoded CRISPR to edit wild populations (theoretical and lab use not public). Edit gene individual —> one copy converts gene inherited from other parent more than 50% of passing it.

Transformation

Insertion of plasmid; pores in membrane created by salts or electricity allow DNA to enter (heat or electric shock)

Transduction (virus)

AAV— DNA inserted but no integrated not passed during cell division & Lentivirus permanently inserts from retroviruses

Transformation vs Transduction

transformation is through holes and modifying physical membrane were as transduction is through virus DNA insertion

Transfection Lipid Particles

Transfection agent surrounds nucleic acid sequences and passes through the cell membrane.

Gene Gun

Compressed gases used to insert DNA into the plants through the cell walls

Transgenic Mice

Use Embryonic stem cells —> modify (transfection/duction) —> incorporate into culture with donor embryo —> chimeric mice mate with wild type —> offspring’s gonads

GMOs/ Bioengineered

transgenic where gene from another organism/ created in lab combined with intended organism

GMOs in Agriculture

Common: crops produce their own pesticide, resistant to herbicides/ diseases
Rare: Crops that can be stored longer and grow better/ more nutritious

Grafting

Have an established root stock then cut off and graft another of species of apples onto the stock. Forming hybrids of two plants by physically attaching them.

Cheese with GMOs

hard cheeses requires an enzyme mix called rennet, 90% of cheese made in US uses these GMO microbes, GMO labelling laws do not require labelings

Synthetic Biology: Fuels

Design GMO microbes which can make useful fuels for cars and electric generators, as well as plastics and other useful materials

Genetic Diseases

Single malfunctioning gene = most are LOF mutations typically recessive

Gene therapy

Treat genetic diseases by replacing or augmenting mutated gene with normal genes (add an addition function gene to recessive LOF).

CONS of gene therapy

Need to modify every cells that’s non functional and can cause other mutation issues/ deletions/ disruption of gene editing, etc…

Outside Body Gene editing

Harvest cells from patient cells —> edited gene inserted into virus —> virus mixing with patients cells —> cells in body are genetically altered. (CRISPR or viral insertion possible)

Successes in Gene Therapy

CRISPR for blood diseases (sickle cell, T-cell leukemia), Retinal diseases, Spinal muscular atrophy (motor neurons loss). No risk of passing mutations to next generation.

Prevent Genetic Diseases

In vitro fertilization & screening, PGD (pre-implantation genetic Diagnosis) can test the embryos before implantation if parents carry genetic diseases.