BIOL 283 - CRISPR ACTIVITY

Describe 2 results from your CRISPR experiment that demonstrated the lacZ gene was cut by Cas9?

-In one plate, blue bacterial colonies turned white through gene editing, indicating that CRISPR-Cas9 was successful. LacZ gene had been made unfunctional. -In another plate, dead colonies (appear as no growth) from the tube wherein HDR was not possible due to lack of arabinose. lacZ gene was cut, but not repaired.

Steps of Cas9 DNA recognition and cleavage

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 DNA three base pairs upstream of the 5'-NGG PAM sequence. 5. The complex releases from the DNA.

Two steps of gene editing

1. Cutting double-strand DNA at a desired location 2. Directing DNA repair to produce a desired sequence change.

three steps of PCR cycle

1. Denaturation 2. Annealing 3. Extension

Briefly explain what happens at annealing step of PCR

62 deg. Celsius for 30 seconds -> temp is lowered so that the primers can attach to the DNA template

Briefly explain what happens at extending step of PCR

74 deg. Celsius for 1 min -> temp. raised and new strand of DNA is made w/ Taq polymerase

Briefly explain what happens at denaturation step of PCR

94 deg. Celsius for 30 seconds -> separate double-stranded DNA into 2 single strands at high heat

Why are bacterial colonies on the starter plates blue?

Bacteria expressing functional B-gal turn blue when they are grown in the presence of X-gal.

What happens when Beta-gal is expressed by bacteria in the presence of X-gal?

Bacteria will turn blue

Why do molecular biologists use the lacZ gene as a target site for inserting DNA sequences?

Bacterial colony color indicates whether or not they were successful. This is called blue-white screening.

Protospacer

DNA region targeted for cleavage by the CRISPR system.

Homology directed repair (HDR)

DNA repair mechanism in which specific proteins patch a double-strand DNA using donor template DNA. 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 the repair.

Non-homologous end joining (NHEJ)

DNA repair mechanism in which specific proteins reconnect the ends of a double-strand DNA break. This process may randomly insert or delete one or more bases that can disrupt gene function or expression.

When you analyze your PCR results by gel electrophoresis, your bands will be visualized using an Ethidium Bromide (EtBr) dye. How will we detect this dye?

Fluoresce bands under UV radiation.

Explain how the differences b/w the IX and IX/ARA starter plates may influence gene editing in the laboratory activity.

IX has no arabinose. IX/ARA has arabinose present, which means the cells have the enzymes needed for HDR. HDR may not be possible in colonies on IX plate.

X-gal

Its hydrolysis by beta-galactosidase yields an insoluble blue pigment and can be used in bacterial cultures to indicate the presence of active beta-galactosidase.

How can cells repair double-stranded breaks in DNA?

Nonhomologous end joining (NHEJ) and homology directed repair (HDR)

primer set designed to detect unmodified lacZ

One primer in the set will bind directly to the Cas9 target cut site. If the target cut site was modified, the primer will not bind. If the target Cas9 cut site was NOT modified, then this primer set will yield a ~1,100 bp amplicon

primer set designed to detect modified lacZ.

One primer in the set will bind to the insert from the donor template DNA. If the target cut site was successfully repaired using the donor template DNA that was introduced to the bacteria, then this primer set will yield a ~650 bp amplicon.

Even in the presence of an sgRNA guide, what DNA element is required for CRISPR-Cas9 target recognition or cutting?

Protospacer adjacent motif (PAM)

What is the purpose of a negative control in a PCR reaction?

Shows if contamination of the PCR with foreign DNA has occurred

What is the purpose of a positive control in a PCR reaction?

Shows that DNA is present but is different from the genomic target sequence

___________ amplifies a specific nucleotide sequence (amplicon) using a single set of _______.

Standard polymerase reaction (PCR); primers

Why is pLZDonor or pLZDonorGuide introduced in the tubes?

The bacteria do not normally produce the sgRNA and donor template DNA required to edit the lacZ gene.

Summary of CRISPR Lab Part 1

Use CRISPR-Cas9 to cut bacterial chromosomal DNA at a specific location within the lacZ gene. Take advantage of cells' ability to perform HDR to cause a desired change in the lacZ sequence. Done by providing cells with large quantities of a donor template DNA.

What happens if the DNA cut is not repaired?

When chomosomal DNA in a bacterial cell is cut, the cell will die.

If the bacteria on the starter plates did NOT have a functional lacZ gene, what color would you expect the colonies to be?

White and opaque

control primer set

Will amplify an unrelated region far downstream of the lacZ gene as a control to verify that chromosomal DNA is present in the sample. If chromosomal DNA was successfully extracted and PCR was successful, whether or not lacZ was modified, then this primer set will yield a ~350 bp amplicon.

Is it possible for repair mechanisms to insert an unexpected sequence even if Cas9 cuts DNA in the correct location?

Yes.

Will cells exposed to arabinose retain the enzymes needed for HDR even if transferred to a plate with no arabinose?

Yes. However, their daughter cells will not produce HDR enzymes unless exposed to arabinose.

chromosome

a DNA molecule with all or part of the genetic material for an organism

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. Cas9 is involved in the natural defense of certain prokaryotes against DNA viruses.

Amplicon

a piece of DNA that was produced through amplification, often by PCR. A PCR product can be called an amplicon

Protospacer adjacent motif (PAM)

a sequence motif immediately downstream of the protospacer sequence in the Cas9 recognition sequence that is required for Cas 9 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

primer

a short sequence of nucleotides (usually 16-24 bases in length) that recognizes a particular sequence of nucleotides on the target DNA sequence;; primers for the polymerase chain reaction are usually synthesized in a laboratory

What can prevent amplification of target PCR sequence?

absence of a target PCR sequence or disruption of primer binding sites

CRISPR was discovered as part of the _____________

adaptive immune system of bacteria

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

In your experiment, the DNA repair mechanism was induced (turned ON) in cells by the addition of what?

arabinose

In E.coli used in the experiment, how is expression of the HDR DNA repair system controlled?

arabinose-inducible promoter; when bacteria are exposed to arabinose, they express/turn on the HDR DNA repair machinery

What happens to bacteria expressing functional beta-gal when grown in the presence of X-gal?

bacteria turn blue

Scaffold region

called the transactivating CRSPR 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

CRISPR

clustered regularly interspaced palindromic repeats; sequences in the genomes of some prokaryotes that act as genomic record of previous viral attack. Along with CRSPR-associated (Cas) proteins, bacteria use the sequences to recognize and disarm future invading viruses. Scientists have adapted this system for genetic engineering purposes.

What makes CRISPR-Cas9 so powerful?

combination of its precision and simplicity

base pair (bp)

complementary nucleotides held together by hydrogen bonds

beta-galactosidase

encoded by the lacZ gene, this enzyme hydrolyzes galactose-containing carbohydrates, including lactose. Conveniently, it also breaks down the colorless compound X-gal into two pieces, one of which goes on to form a deep blue pigment.

Donor template DNA

engineered sequence of DNA required for homology-directed repair in CRISPR gene editing applications; may include a desired sequence flanked on both sides by "homology arms" that match the sequence upstream and downstream of the cut

endonuclease

enzyme which cleaves a polynucleotide chain by separating nucleotides other than the two end ones

pLZDonorGuide

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

pLZDonor

induces 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

Isopropyl Beta-d-1-thiogalactopyranoside (IPTG)

induces transcription of the lac operon

What gene are we seeking to edit in this experiment? How will we determine by visually inspecting the plates next week whether the editing was successful?

lacZ gene; bacterial colonies should appear white & opaque in IX/ARA plates (the one w/ sgRNA). IX plate will not be able to perform HDR.

gene editing

manipulation of genetic material in living cells by adding, removing, and replacing DNA sequences, typically with the aim of altering phenotypes

InstaGene Matrix

microscopic beads that bind divalent cations in solution; the binding or sequestering of divalent cations prevents their availability to enzymes that can degrade the DNA template

Do e.coli bacteria normally produce the sgRNA and donor template DNA required to edit the lacZ gene?

no

Guide RNA (gRNA)

non-coding, short RNA sequence that binds to Cas9 and to complementary target DNA sequences, where Cas9 performs its endonuclease activity to cut the target DNA strand

Plasmids used in CRISPR Day 1?

pLZDonor (control) and pLZDONORGuide

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

lacZ

part of the lac operon in E.coli, this gene encodes the enzyme beta-galactosidase. For decades, molecular biologists have used the lacZ gene as a target site for inserting DNA sequences because the resulting bacterial colony color indicates whether insert was successful.

Arabinose-inducible promoter

promoter that occurs naturally in bacterial systems and that is used in many expression plasmids to allow regulation of expression of a target gene: expression is induced in the presence of arabinose and repressed in its absence

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 cause mutations that can disrupt gene function or expression.

Master mix

the main reagent solution used in PCR, master mix contains all the necessary components (dNTPs, primers, buffer, salts, polymerase, polymerase cofactor)

Polymerase chain reaction (PCR)

the process of amplifying or synthesizing DNA in vitro using primers and cycles of temperature change

multiplex PCR

the simultaneous amplification of multiple amplicons in a single reaction using a unique primer set for each

Multiplex PCR

the simultaneous amplification of multiple targets by polymerase chain reaction. Multiple primer sets are used in the reaction, one for each target