Genetic Engineering: Some Basic Concepts

DNA: The Information Carrier

DNA Replication

Make RNA  Transcription

Make Proteins  Translation

Proteins Do the Work They form cellular structures (such as cell walls, organelles, etc). They regulate reactions that take place in the cell. They can serve as enzymes, which speed-up reactions Everything you see in an organism is either made of proteins or the result of a protein action

Gene Stretch of DNA coding for one protein In Bacteria: a few thousand genes In Humans: ca. 20,000 In Rice: ca. 40,000

Breakthroughs in Bioengineering Sequencing DNA Enzymes that cut DNA at specific locations In vitro synthesis of DNA Cloning: Introducing exogenous genes

Cloning in Bacteria: Easy as π

Examples in Medicine and Industry Insulin Human growth hormone Blood clotting factors Chymosin: enzyme in cheese manufacture (from rennet)

Moving Genes into Plants (I): Ti Plasmid

Moving Genes into Plants (II): Gene Gun

Moving Genes into Animals

Bioenhanced Cattle

Caveats Multiple copies possible No way to control insertion site Insertion into and inactivation of genes

The Ideal: “Surgical” Precision

CRISPR/Cas9 Genome Editing Editing complex includes a DNA-cutting enzyme (Cas9) bound to a short RNA guide strand complementary to a specific genome sequence. The RNA guides the complex to the right sequence; Cas9 makes the cut. Double strand break (DSB) of the DNA follows. Two ways to repair: error-prone at random (left) or specifically by supplying a template (donor DNA) from which the repair system copies the missing piece (right).

Researchers reverse a liver disorder in mice by correcting a mutated gene Nature Biotechnology, March 2014 Mutated gene, unable to metabolize an aminoacid Editing complex includes a DNA-cutting enzyme (Cas9) bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, telling Cas9 where to make its cut. At the same time, the researchers also deliver a DNA template strand. When the cell repairs the damage produced by Cas9, it copies from the template, introducing new genetic material into the genome Use high-powered syringe to rapidly discharge the editing material into a vein. This approach delivers material successfully to liver cells. After ca. 30 days about one-third of all hepatocytes had the edited gene. This was enough to cure the disease Very promising for curing diseases that are caused by single mutations., such as hemophilia, Huntington's disease, and others

“Fixing” Fertilized Eggs Correction of a Genetic Disease in Mouse via Use of CRISPR- Cas9; Cell Stem Cell, Volume 13, Issue 6, (2013) By zygote injection of CRISPR/Cas9, mice or rats carrying desired mutations can be generated in one step Mice with mutations in the Crygc gene or dystrophin gene ( Dmd ) that cause cataracts or Duchenne muscular dystrophy (DMD) can be corrected by coinjection of CRISPR/Cas9 targeting the mutant alleles into zygotes Nevertheless, direct injection of the CRISPR-Cas9 system into zygotes could not produce healthy progeny at an efficiency of 100% and could potentially generate unwanted modifications in the offspring genome, including off-target modifications, which would prohibit its use in the correction of human genetic diseases

Corrections in Germline Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells Cell Research (2015) 25 :67–79 To circumvent these problems, a possible strategy is to correct genetic defects in germline cells, such as Sperm Stem Cells (SSCs), which can be well established from male individuals. Select single SSCs that carry the desired gene modification without any other genomic changes, and use them to produce healthy offspring at 100% efficiency

In Humans? Pre-selection of SSC lines carrying the desired genotype without off-target mutations is feasible. This would enable the generation of healthy progeny, at an efficiency of 100%, from a father carrying a genetic defect Potentially useful in curing (1) male infertility induced by genetic defects, (2) father-carrying dominant disease alleles, and (3) sex chromosome-linked dominant genetic diseases