Genome editing: RNA guided DNA nucleases: CRISPR Cas

Question 1

How does type 2 CRISPR Cas work?

Answer 1

1. Pre-CRISPR RNA generated
2. tracrRNA interacts with repeat
   CRISPR sequences
3. Pre-CRISPR RNA is then cleaved into
   individual fragments called “crRNA”
4. crRNA = tracrRNA + unique CRISPR
   sequence
5. This sequence also contains the
   ADJACENT “PAM” sequence (to bind
   to bacterial PAM)
6. This then forms a complex with Cas9
7. Cas9 then cleaves the foreign DNA with
   the PAM

Question 2

What does PAM stand for?

Answer 2

protospacer adjacent motif

Question 3

How is the CRISPR-Cas9 system made synthetically?

Answer 3

Add Cas9 and single-guided RNA (sgRNA) to target containing PAM sequence

Question 4

How can we introduce new DNA into a target region using HDR (homology directed repair)

Answer 4

1. Eliminate the possibility of NHEJ -
   change specificity of Cas9 nuclease to
   cut DNA on one strand only (“nickase”)
   a. Cut one both stands, at different
   locations
2. Requires two target sequences

Question 5

How can we use defective Ca9?

Answer 5

1. Defective Cas9 (dCas9) cannot cleave
   DNA
2. dCas9 can be “fused to activators or
   repressors” to induce/block expression
   of specific target genes
3. It can also be fused to GFP to monitor
   specific chromosomal locations

Question 6

What are “Homing endonucleases” (HEG) - “selfish genes”?

Answer 6

1. Homing endonuclease genes (HEGs) are 
   typically found between two specific 
   sequences of DNA within the genome.
2. HEG codes for production of an enzymes 
    that recognises these two sequences 
    when they aren't interrupted by the 
    presence of HEG

Question 7

How could homing endonucleases (HEGs) be used to create a hyper functional “gene-drive” or self-perpetuating genetic device that can transmit itself throughout a population at rates that defy mednelian inheritance

Question 7

How could homing endonucleases (HEGs) be used to create a hyper functional “gene-drive” or self-perpetuating genetic device that can transmit itself throughout a population at rates that defy mednelian inheritance

Answer 7

HEGs always inherited by offspring

Question 8

What are the downsides of genome editing?

Answer 8

1. Specificity - “off-targets”
2. NHEJ - innate repair mechanism -
   unwanted changes ; “noise”
3. Ethical considerations