CRISPR

Question 1

What is CRISPR

Answer 1

• clustered regularly spaced palindromic repeated
• CAS = CRISPR associated proteins
• CRISPR and CAS found in 50% of bacterial species and 90% of archea
• first discovered in 1987 but function unknown until 2005 then shown to be a defence mechanism against foreign DNA molecules
• similar to RE but more complex in function
• designed to target specific DNA molecules
• CRISPR-CAs systems have many version but CRISPR-Cas9 focused on primarily

Question 2

CRISPR-Cas9

Answer 2

• Cas genes encode CRISPR-associated proteins
  
  • which have endonuclease activity
• CRISPPR contains unique spacer DNA from invading phases between short 23-47bp palindromic repeat DNA sequences

Question 3

Stage 1: foreign DNA acquisition (aka adaption)

Answer 3

• invading bacteriophage inject foreign DNA into cell
• foreign DNA cut into short segments
• DNA segments are added into bacterial chromosome between repeated palindromic sequences (protospacers)

Question 4

Stage 2: expression

Answer 4

• cas genes are transcribed and translated to Cas proteins
• CRISPR locus transcribed to pre-crRNA
• Pre-crRNA cleaved by CAs proteins and form hairpins and unique short segments of foreign DNA = crDNA

Question 5

Stage 3: interference

Answer 5

• rRNA and trans-activating crRNA are incorporating into Cas protein and allow targeting to DNA sequence that match the unique spacer RNA
• recognize DNA sequence that it has encountered before
• binds and uses Cas endonuclease activity to cleave invading DNA
• presence of protospacer-adjacent motif (PAM) is required in target DNA
• PAM is a short weak consensus sequence

Question 6

Doudna and Charpentier

Answer 6

-in 2012 they showed how CRISPR-Cas9 can be co-opted for genome editing and engineering

Question 7

Genome auditing with CRISPR-Cas

Answer 7

• single guide RNA (sgRNA) replaces natural crRNA and tracrRNA
• sgRNA designed to target a specific sequence in genome
• sgRNA assembles with Cas9 protein to form effector complex
• 20b 5’ portion of sgRNA base pairs to complementary target sequence in genome (8-12b seed is most important
• presence of PAM near seed sequence is essential
• effector complex must bind with PAM then Cas9 unwinds DNA nearby
• if target sequence is present, sgRNA binds with it
• Cas9 makes double stranded cut in genome
• cellular DNA repair mechanisms engaged

Question 8

Cellular DNA repair mechanisms

Answer 8

1. Broken ends can be rejoined without any template
   
   • nonhomologous end joining (NHEJ)
   • frequently results in small insertion or deletions which tend to disrupt gene function
2. Broken ends can be rejoined using a template
   
   • homology directed rejoining HDR
   • template could be the other chromosomal copy, or donor DNA molecules provided by researcher

Question 9

NHEJ

Answer 9

• nonhomologous end joining
• most common type of repair to double strand breaks
• double strand breaks lead to problems with replication, inversions, deletions, duplications, translocations
• no template used
• nucleotides may be randomly inserted or deleted as the cleaved ends of the chromosome are rejoined
• often results in INDELS
• resulting frameshift leads to non-functional alleles —> gene silencing —> knockout

Question 10

Homology directed repair

Answer 10

• HDR
• uses same repair enzymes as in crossing over or recombination
• can use homologous chromosome as template
• in CRISPR experiments, can inject donor DNA at same time as Cas9-CRISPR to stimulate HDR

Question 11

Advantages of CRISPR-Cas9

Answer 11

• relatively cheap and easy
• targeting: can design sgRNA for any sequence
• long recognition sequences of 8-12 nucleotides in seed means more specific targeting then REs
• can use indels created by NHEJ to create gene knockout or determine gene function/phenotype
• can be introduced to intact, living cells
• can introduce Cas9 with donor DNA to stimulate HDR

Question 12

Disadvantages of CRISPR-Cas9

Answer 12

• off-target effects - cleavage sometimes no specific
• depends on cell type
• depends on normal function of repair pathways
  
  • edited Cas9 structure for more complementary binding to DNA, but slower acting
  • Germline cells have enhanced homology-directed repair (repair cleaved alleles by matching homologous chromosomes that were not cleaved
• mosaicism: not all cells edited, different genomes, so get mosaic effect
  
  • common in multicellular embryos
  • delivery of Cas9 not 100% for all cells

Question 13

Gene correction in S-phase injected human zygotes

Answer 13

• tested ability to correct a harmful mutation in pre-implantation human embryos
• injection of CRISPR-Cas9 into normal zygote S-phase caused no harm
• injection into zygotes with mutation resulted in mosaic embryos
  
  • some cells remained untargeted mutant
  • some repaired via NHEJ
  • some repaired via HDR

Question 14

Gene correction in M-phase oocyte vs S-phase zygote

Answer 14

• injection of CRISPR-Cas9 along with mutation carrying sperm during M-phase in oocyte resulted in more successful production of repaired embryos
• most repaired embryos produced by HDR, and some by NHEJ
• use of guide DNA increased proportion of embryos produced by HDR from 68% to 78%

Question 15

Potential uses of CRISPR-mediated genome editing

Answer 15

• basic research (create gene knockouts)
  
  • disrupt genes to determine unknown gene function
• editing genomes to meet human needs/desires
  
  • donor organs from animals
  • improved farm animals
  • domestication of new plants for agriculture
  • de-extinction of extinct species
  • gene drives to eliminate insect-spread diseases

Question 16

Gene therapy and medical interventions

Answer 16

• repair mutations causing human disease
  
  • eg. Mdx mice (animal model of Duchennes muscular dystrophy) which have premature stop codon into exon 23 of dystrophin
  • researchers used viruses to inject CRISPR-Cas9 into muscle cells and edit out DNA with the mutation

-HIV virus removed from human cells

Question 17

Gene Drives

Answer 17

• a DNA construct contains gRNA sequence, Cas9, a payload gene, and flanking sequences (H1, H2)
• once introduced as one copy, it copies itself to homologous chromosome via HDR
  
  • after reproduction, heterozygous offspring are converted to individuals homozygous for gene drive construct
• in this manner, the payload gene can spread rapidly though the population because of non-Mendelian inheritance
• could be used to insert gene for resistance to malaria, or a gene that reduces fertility of mosquitos

Question 18

Genome edited pigs

Answer 18

• a study in 2017 used CRISPR-Cas9 to clip out a section of CD163 that codes for a single protein domain
• the receptor seemed unperturbed, which both of the 2 problematic PRRSV species were blocked from entry into the pigs cells
• the gene edited pigs dont become infected when exposed to the virus
• the enzymes (B-glucanase, xylanase, phytase) break down matter that pigs dont digest
  
  • researchers engineered them to be produced in the modified pigs salivary glands
• the transgenic pigs produced less nitrogen and phosphorus in feces, had a faster growth rate, and boosted feed conversion