Genome editing: induce changes to the sequence of genomic DNA in a controllable manner.
CRISPR: clustered regulatory interspaced short palindromic repeats.
Guide RNA: directs cas9 endonuclease protein to a specific DNA site, each new target only need a new one.
NHEJ features: error-prone, faster, commonly used mechanism.
Homologous recombination: sister chromatin used as template for missing DNA sequence.
HR features: low error rate, slower, cell needs to be in S-Phase.
applications of genome editing: add new sequence of DNA, modify existing dna sequence, delete DNA, inactivate a protein-coding gene.
chimer guide sgRNA: gRNA and tracrRNA.
Limitations of Crispr: off-targets, limited by PAM site, large protein so not easy package as virus delivery.
CRISPR/Cas 9 double nickase system: reduces off targets
dCAS9: a mutant cas9 protein that cannot cut DNA, used for targeting fused transcriptional activation or repression domains, without editing DNA.
Base Editors: used for single point mutations that don't cut the DNA.
BEs: fusions of dcas9 with a base-modifcation enzyme that operates on ssDNA, but not dsDNA.
R-loop: base pairing between the gRNA and target DNA leads to displacement of a small segment of ssDNA, bases within this ssDNA bubble are modified by the deaminase enzyme.
Agriculture Genetic engineering: Insert genes or modify genes to create desirable traits.
Molecular cell biology GE: Modify genomes of human cell lines for biomedical research.
Animal models GE: Much faster way of creating transgenics, disease models, knockout mice
Gene Drives :Used for population control of pests eg. mosquitoes
Gene Drives: insert genes encoding crispr machinery and accompanying ‘payload’ gene into DNA.
Clinical appliactions GE: Cell therapy, gene therapy.
Germline Editing: passed onto all cells in baby including germ cells that go on to make further generations.