Genetic engineering

Genes get moved within or between chromosomes Naturally: __________ ______ cuts and moves genes (transposons) E.g., pigment genes of Indian corn Artificially: ________ fragments Genes are moved with restriction enzyme tools E.g,. Human _____ gene into bacteria Engineering > gene transplants

Cut and Paste technology ________ enzymes: DNA cutting tools Normal function: attack foreign ____ “Restrict” foreign DNA by ______________ Cut at a specific __________ sequence on DNA: cut v CGAATTCA GCTTAAGT segment of DNA Restriction sequence Sketch 1

Cutting creates Restriction _________ Fragments have single-stranded ‘_________’ fragments sticky ends AATTCA GT CGAATTCA GCTTAAGT cut GCTTAA CG Sketch 1

Fragments can be ‘______’ onto other strands of DNA Must cut “target” DNA with ____ restriction enzyme Makes sticky end of target ___________ AATTCA GT fragment TTTT GCTTAA AAAACG “Target” DNA Sketch 2

Sticky ends hydrogen-bond readily AATTCA GT TTTT GCTTAA AAAACG Sketch 2

Sticky ends hydrogen-bond readily AATTCA GT TTTT GCTTAA AAAACG Hydrogen bonds Sketch 3

But sugar-phosphate backbones remain un-bonded Bonded with DNA ______ Resulting DNA strand = __________ DNA A combination of DNA from multiple _______ AAAACG AATTCA GT TTTT GCTTAA Sketch 4

C.R.I.S.P.E.R. DNA editing technology (Clustered Regularly-interspaced Short Palindromic Repeats) “The most powerful biological tool ever known” Works like a word processor “_____________” function Much cheaper and faster than existing techniques First used to edit human genes in 2014 Will soon cure genetic diseases, including cancer But opens the door for “designer” ______

Bacterial origins of CRISPER Basic mechanism was discovered in E. coli ______ Bacterial defense against _______ Bacterium stores bits of _____ DNA in its own DNA Allows it to _______ and destroy a virus Human immune system has a similar mechanism “B” lymphocytes store bacterial surface ______ If bacterium, is re-infected: ___ genes make 2 kinds of proteins (enzymes): - ______ to unzip viral DNA - _______ to cut (destroy) viral DNA of the virus If viral DNA is a new type: Cas proteins ____ viral DNA ____ bacterial DNA

CRISPER Cas9 gene editing system Cas9 protein CRISPER Cas9 gene editing system protein Nuclease “scissors” tracrRNA ____ crRNA in place crRNA ____ for target sequence

Function: 1) Introduce DNA with “target” sequence specific to Cas9 2) Target aligns with crRNA 3) Cas9 nucleases cut target Use: 1) Replace crRNA-trcrRNA complex with gRNA “_____ RNA”, ___-made for a given task Has code for whatever gene you want to edit 2) Cas9 then cuts out the target gene sequence 3) Additional enzymes can _______ that gene with something else E.g., better form of the gene; type of gene ______

Older types/uses of genetic engineering 1. Gene sequencing: To determine the ________ sequence of genes Uses: Identify ________ versions of a gene Determine entire __________ of organisms: Gemone of many species has been sequenced 2. DNA Fingerprinting Uses: distinguish between unique genetic _____ E.g., between individuals, species, or larger taxa Individuals have slight differences in genetic code E.g., alternate _______

3. Transgenic organisms Contain genes from other sources E.g., implanting desirable traits in crop plants Certain bacteria naturally implant genes into plants E.g., _________ causes tumors in plants Technique: a. Add a gene to such a bacterium’s DNA b. Allow bacterium to implant gene into plant DNA Natural ‘___________’ DNA c. “_______” cells from this plant Each cell becomes a plant – a _____

Currently, genetically modified (GM) crops contain: ___________ proteins E.g., ____ protein from Bacillus bacteria Damages gut lining of ______ _________ proteins strawberries Anti-freeze proteins E.g., isoform HPLC 6 -isolated from _____ -injected with Agrobacterium

___________ resistance Allows heavy spraying against weeds ________-ready soybeans (glyphosate)

Transgenic animals: Faster growth Larger body size More efficient ______ use (lbs. of flesh / lbs. of food)

Political opposition to GM foods: Many nations now label or ban ‘GM’ foods (called ‘_____________’ by some) Consider unknown risks too high E.g., recombined genes might disperse in _____ Might “_____” to related species