Difference between revisions of "20.109(S22):M2D3"

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==Protocols==
 
==Protocols==
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===Part 2: Research sgRNA expression plasmid===
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After identifying the sgRNA_target sequence that is best for targeting the gene of interest in the host genome, the sgRNA_target is inserted into an expression plasmid.  This is achieved using primers and PCR in the procedure described in Part 2.  Before you review the approach used to insert the sgRNA sequence, first familiarize yourself with the important features of the expression plasmid.  The expression plasmid used for regulating gene expression via the CRISPRi system is described in the following article:
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Larson ''et al.'' "[[Media:CRISPRi NatMeth2013.pdf|CRISPR interference (CRISPRi) for sequence-specific control of gene expression.]]" ''Nature''. (2013) 8:2180-2196.
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In this exercise, you will explore the features present in the plasmid that are necessary to express the sgRNA_target sequence (see plasmid map below).
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[[Image:Fa20 M3D2 sgRNA expression plasmid features.png|right|500px|thumb|Image modified from Larson ''et. al.'' ''Nature''. (2013) 8:2180-2196.]]<font color =  #4a9152 >'''In your laboratory notebook,'''</font color> complete the following:
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*Review the details provided in the caption for Fig. 1B of the Larson ''et. al.'' article.
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*Describe the purpose / role for each of the following features that are present in the bacterial sgRNA plasmid.  Please note: you many need to reference resources outside of the article!
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**Constitutive - pJ23119
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**Base-pairing region
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**dCas9 handle
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**''S. pyogenes'' terminator
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**Term (rrnB)
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**AmpR
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**ColE1
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**+1
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**restriction enzyme recognition sequences: EcoRI, BglII, and BamHI
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====Synthesize sgRNA_target sequence====
 
====Synthesize sgRNA_target sequence====

Revision as of 14:22, 21 January 2022

20.109(S22): Laboratory Fundamentals of Biological Engineering

Sp17 20.109 M1D7 chemical structure features.png

Spring 2022 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       M1: Drug discovery        M2: Metabolic engineering        M3: Project design       


Introduction

Protocols

Part 2: Research sgRNA expression plasmid

After identifying the sgRNA_target sequence that is best for targeting the gene of interest in the host genome, the sgRNA_target is inserted into an expression plasmid. This is achieved using primers and PCR in the procedure described in Part 2. Before you review the approach used to insert the sgRNA sequence, first familiarize yourself with the important features of the expression plasmid. The expression plasmid used for regulating gene expression via the CRISPRi system is described in the following article:

Larson et al. "CRISPR interference (CRISPRi) for sequence-specific control of gene expression." Nature. (2013) 8:2180-2196.

In this exercise, you will explore the features present in the plasmid that are necessary to express the sgRNA_target sequence (see plasmid map below).

Image modified from Larson et. al. Nature. (2013) 8:2180-2196.
In your laboratory notebook, complete the following:
  • Review the details provided in the caption for Fig. 1B of the Larson et. al. article.
  • Describe the purpose / role for each of the following features that are present in the bacterial sgRNA plasmid. Please note: you many need to reference resources outside of the article!
    • Constitutive - pJ23119
    • Base-pairing region
    • dCas9 handle
    • S. pyogenes terminator
    • Term (rrnB)
    • AmpR
    • ColE1
    • +1
    • restriction enzyme recognition sequences: EcoRI, BglII, and BamHI


Synthesize sgRNA_target sequence

Fa20 M3D2 full sgRNA schematic.png
In the previous laboratory session you learned how to best target genes of interest by designing sgRNA sequences that bind within the host genome. The sgRNA sequence is what enables the CRISPRi system to specifically target the promoter or coding region of a gene, and thereby regulate gene expression. Thus far, we have described the sgRNA as only containing sequence complementary to the targeted gene in the genome, but this is only half of the full sgRNA used in CRISPR-based technologies. The second half of the sgRNA is a 'handle' that binds dCas9 (or Cas9 in the native CRISPR system). As shown in the image to the right, the targeting sequence and the dCas9 handle together compose the sgRNA. To synthesize the full sgRNA, the basepair sequence was submitted to IDT-DNA, a commercial company that specializes in DNA synthesis chemistry. To generate the full sgRNA, the basepair sequences for the targeting sequence and the dCas9 handle were simply submitted as a single DNA strand.

Before we continue, we should review the process used to generate actual primers that can be used to amplify DNA. Current oligonucleotide, or primer, synthesis uses phosphoramidite monomers, which are simply nucleotides with protection groups added. The protection groups prevent side reactions and promote the formation of the correct DNA product. The DNA product synthesis starts with the 3'-most nucleotide and cycles through four steps: deprotection, coupling, capping, and stabilization. First, deprotection removes the protection groups. Second, during coupling the 5' to 3' linkage is generated with the incoming nucleotide. Next, a capping reaction is completed to prevent uncoupled nucleotides from forming unwanted byproducts. Lastly, stabilization is achieved through an oxidation reaction that makes the phosphate group pentavalent. For a more detailed description of this process, read this article from IDT DNA.

Insert sgRNA_target sequence into expression plasmid

The Q5 Site Directed Mutagenesis Kit from NEB was used to insert the sgRNA_target sequence into the expression plasmid. For this, a reaction was prepared with the following: the sgRNA_target primer, a universal CRISPRi primer, and the expression plasmid.

Image modified from Larson et. al. Nature. (2013) 8:2180-2196.
As shown in the figure to the right, the steps used to insert the sgRNA_target sequence into the expression vector are based on the principles of PCR. The expression plasmid is the template in this amplification reaction. For amplification to occur, a forward primer and a reverse primer are required. In this reaction, the forward primer (labeled Primer Ec-F in the figure) is the full sgRNA_target that consists of the targeting sequence and the dCas9 handle. The dCas9 handle part of the primer anneals to the complementary sequence in the expression plasmid. The targeting sequence does not anneal to the expression plasmid, instead this part of the primer is incorporated into the amplification product during PCR. The reverse primer (labeled Primer Ec-R in the figure) anneals to a complementary sequence in the expression plasmid and reads away from the forward primer. The product from each of these primers is a single-stranded DNA molecule. Because these single-stranded products are complementary, the strands will anneal and form a double-stranded product.

In your laboratory notebook, complete the following:

  • Draw the single-stranded product (5' - 3') that is generated from the forward primer. Draw the product generated from the reverse primer.
    • Include the features that are in each product.
  • The single-stranded products will anneal to form a double-stranded product. Is this double-stranded product linear or circular? Why?

A more technical depiction of the protocol used to insert an sgRNA_target sequence into the expression plasmid is included below. Briefly, in Step 1 DNA polymerase copies the plasmid using the sgRNA_target primer to insert the target sequence. Following PCR amplification the product is a linear DNA fragment. In Step 2 circular plasmids that carry the sgRNA_target sequence are generated when the double-stranded DNA is phosphorylated (Step 2A) and then ligated (Step 2B). Following the amplification reaction, the expression plasmid template that does not contain insert is present in the reaction product. To ensure that only the sgRNA_target-containing expression plasmid is used in the next steps, the parental DNA is selectively digested using the DpnI enzyme (Step 2C). The underlying selective property is that DpnI only digests methylated DNA. Because DNA is methylated during replication in host cells, DNA that is synthetically made via an amplification reaction using PCR is not methylated. Lastly, in Step 3 the sgRNA_target-containing expression plasmid is transformed into competent cells that propagate the plasmid.

Schematic for inserting sequences into plasmids using SDM technique. Image modified from Q5 Site-Directed Mutagenesis Kit Manual published by NEB.

Reagents list

==Navigation links==