19. Crispr Cas9 - Lecture notes 19 PDF

Preview

Summary

Dr. Huwe for ACHS post-bac genetics. Exam 3 material. All exams are cumulative...

Description

CRISPR Cas9  CRISPR = clustered regularly interspaced short palindromic repeats  Used for gene editing purposes, 1st identified in E. Coli, has 2 parts: o Repeats: short sequence of DNA that is palindromic o Spacer DNA: non-identical/unique portions of DNA that are interspaced between the repeat regions  Spacer DNA matches up to the viral DNA (from old infections)  Genes associated with CRISPR = Cas genes  Cas proteins (helicases/nucleases = unwind/cut DNA)  Process without immune system: bacteriorphage injects its DNA  DNA will hijack the cell and imbed into the genome  makes a bunch of bacteriophages  cell death  Process with CRISPR system: will transcribe/translate proteins and transcribe DNA  crRNA (a way to fight viral DNA by breaking it apart)  immune system doesn’t allow for infection and destroys it o If it injects DNA that doesn’t have a correct spacer that matches  will create a Cas1 protein (takes DNA and breaks it apart)  will copy and insert it into the CRISPR system  Scientists believed they could hijack the CRISPR system in order to inactivate genes or imbed new genes o Discovered CRISPR/Cas9 system in streptococcus pyogenes  Only contained 1 protein, Cas9 – contained a nuclease to cut DNA in 2 places  Created 2 strips of RNA: crRNA (fits into the Cas) and tracrRNA (holds the crRNA in place in the Cas) o They created the trcrRNA-crRNA chimera [gRNA – guideRNA] (new type of RNA)  gRNA: contains info on where to cut  Cas9: protein that actually does the cutting o To insert a new gene, need 3 parts: Cas9, gRNA and new host DNA to insert



Cas9 Protein: o Inside is a section of RNA that is attached to the protein (can be specific for whatever gene)  it will search along the DNA until it finds the complementary strand and then halts  Cas9 protein has endonuclease activity to cut the DNA, 2 ways:  1. If you leave the cut strand by itself  non-homologous end-joining (NHEJ)  will cause mutations and break the function of whatever gene it is  Bacteria uses this method to try and kill the virus  2. If you include a chunk of DNA that is complementary to the gene of interest  homology directed repair (HDR)  can include new genes into it  Uses as a template for repair



Another Cas system involves nicking only a portion of the DNA instead of endonucleases activity



Different vectors used to get genetic info into the system o Retrovirus: DNA is inserted into the host genome, can have random insertion  cancer or break other important genes o Adenovirus: able to target non-dividing cells, may have some host-immune reactions o AAV (adeno-associated virus): has a smaller capacity for the amount of genes that can be inserted but less of a chance of an immune response o Synthetic vectors: could contain the already made protein inside the vesicle o Non-viral vectors have good advantages but typically have a low efficiency...