Genetic engineering and CRISPR (16)

Genetic engineering

manipulate genetic makeup of an organism

Why would u manipulate DNA

Alter the genome of the organism to suit the purpose

novel DNA molecules

learn when and where gene expressed

A large amount of proteins made using expression vector

Artificial selection examples

Dog breeds, corn, recombinant DNA, Louise brown (ivf baby), sheep clone, mitochondrial replacement therapy

Plasmid cloning

Early version, still valuable but many variants developed

restriction enzyme, bacterial host, plasmid vector

plasmid cloning process

double-stranded expression w/ promoter sequence, restriction nuclease + insert protein coding DNA sequence, introduce recombinant DNA into cells

Plasmid cloning examples

insulin, growth hormone, factor 7

Site directed mutagenesis

Changing the sequence of expressed gene by altering DNA sequence,

Learning how a genome does what it does (single gene, isolate a gene, change it, clone it into a vector

Insert vector into an organism and see what differences in phenotype altered gene produces

transgenic organisms

new gene to genome > done via replacement, knockout, or addition

haploid organisms are easily accomplished through homologous recombination

Genetic engineering in complex organism

Creating transgenic organisms is more difficult in more complex diploid organisms, has to be inserted into germ line, genome editing using homologues recombination is possible, involved and time consuming

ZFNS

zinc finger recognition domain to target DNA,

Transcription activator-like effector nucleases (TALENS)

based on transcription-activating proteins from the plant pathogen Xanthomonas

both required the construction of a new protein for each new target

DNA engineering in complex organism

Creating transgenic organisms is harder in diploids

inserted into germline

pretty hard untill CRISPR

CRISPR

clustered regularly interspaced short palindromic repeats found in prokaryotes, act as an immune system, can be used in eukaryotes as well, creating animal models of human disease, gws of humans, genetically modifying plants, and human cells

Crispr methodology in immune system

bacterium infected by phage, Cas proteins bind the phage DNA + chop it up, some pieces are retained and stored between palindromic repeats, serving as memory which can trasncribe the piece and target that phage when it invades

what is cas9

cas9 targets dsDNA sequence making it useful for eukaryotic cells

cas9 mechanism

cas9 guided to its target dsDNa by CRISPR- cas ribonucleoproteins (cRNPs)

CAS9 creates db break in DNA

A single precise blunt-end cleaves 3 nucleotides upstream of the proto-spacer adjacent motif (PAM)

CRISPR-cas9- gene knockout

Cas9 only breaks the DNA; it does not introduce new DNA

Good for knocking out, the broken DNA is repaired inefficiently via non-homologous end joining (NHEJ) usually deactivates the gene ( causes frameshift )

CRISPR-cas9- gene replacement

homologous recombination, simultaneously introduces DNA that has flanking sequences that match sequences flanking the break

New CRIPSR application

activation-repression, base editing, epigenetic modifcation

whole genome screening

in silico gRNA design > synthesized gRNa oligos > CRIPSR library > amplified CRISPR library> lentivirus containing CRISPR library, Cas9 expressing cells> infect cells w/ library > apply ± selection and identify hits with NGS

CRISPR and RNAi

are logically similar but NOT homologous

how does CRISPR resemble RNAi

both systems use a guide RNA to target a specific nucleic acid sequence using Watson-crick complementarity

CRISPR differences from RNAi

CRISPR cuts db dna

RNAi cuts RNA (other RNAi-related mechanisms can direct DNA methylation or block mRNA translation)

Gene editing applications

Laboratory tools, new antibiotics, edit genes for disease/gene therapy, edit crops for disease or drought resistance; increases nutrition, eliminate disease carrying vectors- gene drive

Designer babies

First human gene-editing treatment

Hunter syndrome, mutation in iduronate-2-sulfatase (IDS), ZFNs introducing correct IDS to liver cells, 2 patients with medium dose showed improvement

Application of gene drive

Malaria: kills 1 million people, 90% of deaths in young children, modified mosquitoes can not transmit plasmodium, Release 1200 into a cage w/ malaria infected mice and 1200 unmodified mosquitos, within 9 genes modified mosquitos became majority thru gene drive

normal inheritance vs gene drive inheritance

Altered gene and wt. > only 50% chance of passing on genes

Gene drive inheritance > altered genes as gene drive, one copy converts gene inherited from other parent, more than 50% chance of passing it on

General global opinion on genetic engineering

Most would not alter their baby to make it smarter, most would think its appropriate to remove disease gene

Crispr baby

illegal, in China,

Downsides: could be off-target effects: deleting CCr5 has other disease implications

Upsides: aids free?