CRISPR

Question 1

CRISPR IMMUNE SYSTEM

Answer 1

1. Bacteriophage injects its viral DNA into bacterium
2. Cas enzymes chop up the bacteriophage DNA and insert a copy into their CRISPR array (as spacer DNA, in amongst the palindromic repeats)
3. When the CRISPR array is transcribed, tracr and pre-CRISPR RNA are created, and Cas9 is transcribed and translated
4. tracRNA, pre-CRISPR RNA and Cas9 enzyme combine to create an active Cas9 complex
5. If reinfected by the bacteriophage, a Cas9 enzyme with the complementary RNA sequence will recognise and bind to the PAM on the bacteriophage DNA and cut DNA a few bases upstream of PAM

Question 2

sgRNA Process

Answer 2

1. sgRNA is designed to have complementary sequence to target DNA
2. sgRNA is combined with a Cas9 enzyme to create an active Cas9 complex
3. Cas9 complex is inserted into cells of interest
4. Cas9 will recognise and bind to PAM downstream of target DNA and cleave the target DNA a few bases upstream of PAM
5. Cell will try and repair this cut in DNA and will likely create a mutation in the process, inactivating the gene. OR scientists can insert a gene of interest within the cut DNA to knock in a gene.