Biol 315L Final CRISPR

• What does C.R.I.S.P.R. stand for?

  Clustered regularly interspaced palindromic repeats
• What is CRISPR?

  Sequences in the genomes of some prokaryotes that act as a genomic record of previous viral attack
• Along with CRISPR associated (Cas) proteins, bacteria use the CRISPR sequences to recognize and disarm future invading viruses.
• CRISPR-Cas9 is not the first programmable gene-editing tool, nor is it necessarily the most precise. Other gene-editing tools, like TALENs or zinc-finger nucleases, are also programmable and precise, but they are very expensive and laborious to use. What makes CRISPR-Cas9 so powerful is the combination of its precision and simplicity.
• The CRISPR-Cas9 system consists of the following components

  Cas9 enzyme (Cas9)

  Single guide RNA (sgRNA)

  Protospacer adjacent motif (PAM)
• Cas9 enzyme (Cas9)

  A bacterial endonuclease that forms a double-strand break (cuts) DNA at a specific site within a larger recognition sequence, or target site. The Cas9 recognition sequence includes a 20-nucleotide sequence called the protospacer that is determined by a guide RNA bound to the enzyme
• Single guide RNA (sgRNA)

  An engineered form of guide RNA that forms a complex with Cas9. The sgRNA is an approximately 100 nucleotide–long fusion of two regions that occur as separate RNAs in nature:

  • Guiding region

  • Scaffold region
• Single guide RNA (sgRNA) - Guiding Region

  Part of the CRISPR RNA or crRNA in nature, a typically 20-nucleotide region that is complementary to the target DNA sequence and that defines where Cas9 cuts. Scientists can easily customize this sequence for their own targets
• Single guide RNA (sgRNA) - Scaffold Region

  Called the transactivating CRISPR RNA or tracrRNA in nature, a region that forms a multi–hairpin loop structure (scaffold) that binds tightly in a crevice of the Cas9 protein. The sequence of this region is typically the same for all sgRNAs
• Protospacer adjacent motif (PAM)

  A sequence motif immediately downstream of the protospacer sequence in the Cas9 recognition sequence that is required for Cas9 function. Cas9 recognizes the PAM sequence 5'-NGG where N can be any nucleotide (A, T, C, or G). When Cas9 binds the PAM, it separates the DNA strands of the adjacent sequence to allow binding of the sgRNA. If the sgRNA is complementary to that sequence, Cas9 cuts the DNA
• Steps of CRISPR

  1. Cas9 binds an sgRNA.
  2. The Cas9-sgRNA complex binds to a PAM site on the target DNA.
  3. The guiding region of the sgRNA binds to the target DNA sequence.
  4. Cas9 makes a double-stranded break in the DNA three base pairs upstream of the 5'-NGG PAM sequence.
  5. The complex releases from the DNA.
• 1. Cas9 binds an sgRNA.

  Cas9 recognizes and binds the scaffold (tracrRNA) region of an sgRNA. The nucleotide sequence of the scaffold region determines its structure, which is tailored to fit within the Cas9 protein like a key fits into a lock.
• 2. The Cas9-sgRNA complex binds to a PAM site on the target DNA.

  Cas9 requires a particular PAM sequence (5'-NGG) to be present directly adjacent to the protospacer sequence. When the Cas9-sgRNA complex recognizes and binds a PAM site, it separates the DNA strands of the adjacent protospacer sequence to allow binding of the sgRNA.
• 3. The guiding region of the sgRNA binds to the target DNA sequence.

  The guiding region of the sgRNA attempts to base-pair with the DNA. If a match is found, the process continues. Otherwise, the complex releases and attempts to bind another PAM and target DNA sequence.
• 5. The complex releases from the DNA.

  The Cas9-sgRNA complex releases the cut DNA and is ready to repeat the process.
• The lacZ gene codes for the enzyme b-galactosidase (b-gal).
• The lacZ gene is part of the lac operon, a collection of genes that allows bacteria to use lactose, a milk sugar, as a food source.
• b-gal breaks down the colorless compound X-gal into two pieces, one of which is deep blue.
• If b-gal is expressed by bacteria in the presence of X-gal, they will turn blue.
• Gene editing involves two steps:

  Cutting double-strand DNA at a desired location and then directing DNA repair to produce a desired sequence change.
• When chromosomal DNA in a bacterial cell is cut, the cell will die unless it’s able to repair the cut.
• Cells can repair double-stranded breaks in DNA in several ways, including:

  • Nonhomologous end joining (NHEJ)

  • Homology directed repair (HDR)
• Nonhomologous end joining (NHEJ)


  specific proteins reconnect the ends of the double stranded break back together. This process may randomly insert or delete one or more bases and can cause mutations that can disrupt gene function or expression
• Homology directed repair (HDR)

  enzymes patch the break using donor template DNA, which is required for HDR. Researchers design the donor template DNA, which may include a desired sequence flanked on both sides by “homology arms” that match the sequence upstream and downstream of the cut. A complementary DNA strand is created during repair
• A gene in the lac operon, lacZ encodes an enzyme called b-galactosidase (b-gal), which catalyzes the hydrolysis of the sugar lactose into its component sugars. b-gal can also hydrolyze a sugar analog called X-gal, which produces a blue pigment after it is hydrolyzed. Bacteria expressing functional b-gal turn blue when they are grown in the presence of X-gal.
• pLZDonor

  (control) includes a donor template DNA sequence that will be used by the HDR machinery to fix double-stranded DNA breaks. The donor template DNA includes an insert sequence, which will be inserted into the lacZ gene and impair its function
• pLZDonorGuide

  includes both the donor template DNA sequence from pLZDonor and a sequence that codes for the sgRNA. Once transcribed, the sgRNA will direct Cas9 where to cut lacZ
• HDR


  Homology Directed Repair
• HDR Requirements

  Donor template and repair enzymes
• IX/ARA


  Has Arabinose and HDR
• IX

  Does not have Arabinose - so no HDR
• Bacterial Plasmid


  •Not part of bacteria’s genome

  •Contain small number of genes

  •Can replicate on their own

  •Horizontal gene transfer between bacteria
• Bacterial Transformation Steps:

  1. Competent cell preparation
  2. Transformation
  3. Recovery
• Bacteria with a functional lacZ gene are

  blue
• Bacteria with a disrupted, nonfunctional lacZ gene are

  white
• Incubate the plates upside-down at 37°C for 24 hours or at room temperature for 2–3 days.
• IX plate

  IPTG/X-gal plate
• IX/ARA plate

  IPTG/X-gal/Ara plate
• pD

  pLZDonor plasmid
• pDG

  pLZDonorGuide plasmid