Recombinant DNA Technology

Recombinant DNA Technology

• Enables the mixing and matching of specific DNA sequences from any organism
• Creates DNA molecules not found in nature
• Is a cornerstone of biotechnology
• Involves engineering of genes, cells, and organisms for basic research

History

• Manipulating genes using technology is termed genetic engineering or recombinant DNA technology (rDNA)
• Peter Lobban, as a graduate student with A. Dale Kaiser at Stanford, proposed the recombinant DNA technique
• Recombinant DNA technology was enabled by the discovery, isolation, and application of restriction endonucleases
• Werner Arber, Daniel Nathans, and Hamilton Smith received the 1978 Nobel Prize in Medicine for restriction endonucleases

Process of Recombinant DNA Production

• Begins with the isolation of a gene of interest, which is then inserted into a vector and cloned
• A vector, which can independently grow, is typically either a bacterial plasmid or a viral phage
• The gene of interest (foreign DNA) is integrated into the plasmid or phage, which is then called recombinant DNA
• Cloning is necessary to produce multiple copies of the DNA since the starting amount is insufficient for insertion into host cells
• Vectors can be introduced into host cells like mammalian, yeast, or special bacterial cells for protein expression
• The host cells then synthesize the foreign protein from the recombinant DNA
• The foreign or recombinant protein can be isolated and purified in large amounts when the cells are grown in vast quantities

Applications

• As genetic diseases become more prevalent and agricultural land decreases, recombinant DNA is increasingly important
• Enhances crop production, focusing on drought- and heat-resistant varieties
• Used in creating recombinant vaccines, such as those for Hepatitis B
• Important in preventing and treating sickle cell anemia and cystic fibrosis
• It helps in the production of clotting factors, insulin, recombinant pharmaceuticals and generating plants that produce insecticides, as well as somatic gene therapy

Restriction Enzymes

• Restriction enzymes (REs) allow cutting of specific DNA pieces
• Cutting DNA is a step required to isolate DNA fragments
• Each RE recognizes and cuts at a specific restriction site
• Cuts are commonly asymmetrical
• Similar ends of cut DNA can pair together, then be sealed, or ligated, by DNA ligase

Cloning Vectors

• Restriction Enzymes help stitch together fragments of useful DNA into recombinant molecules
• Recombinant molecules can be used to "clone,” or make many copies, of a bacterial gene of interest
• Vectors are used to insert a recombinant DNA molecule into a recipient host bacterial cell
• Example vectors: Plasmids, Phages and Cosmids

Plasmid Cloning Vectors

• Fragments from two plasmids with antibiotic resistance genes can be cut using the same RE and then ligated with DNA ligase
• Strains exhibit traits from both plasmids after inserting the recombinant plasmid into bacteria
• Common plasmid vectors include: origin of replication, selectable marker gene, multiple cloning site, small size and high copy number

Method of Gene Cloning

• Identification and isolation of the desired gene or DNA fragment to be cloned
• Insertion of the isolated gene in a suitable vector
• Introduction of this vector into a suitable organism/cell called host (transformation)
• Selection of the transformed host cells
• Multiplication/expression/integration followed by expression of the introduced gene in the host

CRISPR-Cas System

• CRISPR-Cas-based genome editing began in 1993 with the study of a salt-tolerant archaeon
• Included short repeating DNA sequences separated by spacers with no similarity to each other or any other sequences in the genome
• In 2005 researchers found that spacers were DNA of different viruses that could infect cell-providing immunity against future infections
• The Clustered Regularly Interspersed Short Palindromic Repeat locus is transcribed into long pre-crRNA (CRISPR RNA), later processed into shorter RNA fragments
• These fragments contain a spacer region with an RNA copy of part of a viral genome flanked by short stretches of bacterial encoded repeat sequences
• The Cas protein's crRNA binds to complementary DNA sequences of invading viruses
• crRNAs are base paired with tracrRNA and bound to CRISPR-associated protein (CAS protein)
• Cas proteins are endonucleases, cutting DNA complementary to crRNA and can be guided via complementary crRNA to specific genome sequences

CRISPR-Cas System for Genome Editing

• Chemically synthesized DNA is transcribed in vitro to create a fusion of crRNAs and tracrRNAs, producing a single guide RNA (sgRNA)
• The sgRNA is mixed with plasmid DNA that contains Cas9
• Introduction of this mixture into a cell expresses Cas9
• The sgRNA guides Cas9 to complementary target sequence and the complex makes double-stranded cuts in DNA

Genome Editing and Repair

• DNA repair mechanisms are used for genome editing using CRISPR-Cas system
• Cells can repair double-strand breaks using homologous recombination
• CRISPR-Cas based genome editing is the next revolution in biology

CRISPR-Cas Genome Editing in Agriculture and Domestic Animals

• CRISPR-Cas genome editing in animals and crops: gene-modifying that produces GM crops or animals do not leave bits of foreign DNA in the genome
• Three genome-edited agricultural products on the market include: Mushrooms that resist browning, extra-oil producing crop plant (Camelina sativa) and drought resistant and salt tolerant soy beans
• Genomes of domestic animals also altered using CRIPSR-Cas, like pigs resistant to porcine respiratory and reproductive virus (PRRSV)
• CRISPR-Cas edited organisms are not regulated and the USDA has determined that crops and livestock that do not contain foreign DNA are not substantially different

Gene Drive

• A genetic engineering process using CRIPR-Cas to drive a particular form of gene throughout a population in just a few generations
• Initially heterozygous organisms become homozygous for the altered allele
• Mating between mosquitos homozygous for engineered allele and wild-type mosquito results in the entire population containing only the modified form of gene

Gene Therapy

• Provides an approach to treating or curing diseases that involve modifications in the genome
• The disease must be due to defects in a single gene, the sequence of the normal allele must be known, and a method must be available to introduce the allele into affected individuals
• It must be expressed in correct tissues, in the correct amount, and at the correct time

Gene Therapy and CRISPR

• CRISPR-Cas9 system is used in cases of dominant disease alleles to replace defective allele with functioning one
• Therapeutic gene delivery uses engineered viruses (vectors) to transport genes into human cells
• Viral genomes are changed to incorporate therapeutic genes

Approaches to Gene Therapy

• Ex vivo gene therapy involves removing cells that require the therapeutic gene, infecting them with a viral gene therapy vector, and integrating the therapeutic gene into the patient's genome
• In vivo gene therapy uses a viral gene delivery vector that is injected into bloodstream and transported throughout body