Genetics Lab report

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LAB 5:

CRISPR OVERVIEW and DESIGNING GUIDE RNA OBJECTIVES

1. Identify and describe the basic elements of the CRISPR/Cas system in Bacteria.

2. Define gene silencing.

3. Describe the roles of the target DNA, gRNA, Cas9, and plasmids in the CRISPR

gene editing experiment in yeast.

4. Use genetic databases to generate gRNA, and to learn about target genes.

BEFORE LAB 1. Read the lab handout before lab. If you do not prepare adequately for lab, you

will not be able to complete the lab in the time allotted.

2. Watch the video on CRISPR (also posted on D2L):

https://www.youtube.com/watch?v=MnYppmstxIs

3. Complete the pre-lab quiz before coming to lab.

GENERAL NOTES ON LAB • You will work in groups of 2 students. You may divide up the activities within

your group, as long as each student masters all of the skills and concepts. Be

sure to ask questions if you are unsure of any instructions.

• Take careful notes as you do the lab. You will need these notes to write your final

paper at the end of the semester.

• You will turn in this lab handout for a grade. Be sure you have all

questions answered in the handout.

CRISPR LABS OVERVIEW This series of labs will occur over eight weeks. In these labs, we will be targeting and

silencing genes in fission yeast (Schizosaccharomyces pombe) and detecting the

phenotypes of the modified yeast.

Your group will present your findings in an oral presentation at the end of the semester,

and each student will turn in a written paper describing the experiment and its findings.

Therefore, you should take very careful notes during each lab, so that you can

address any and all questions about the experiment at the end of the semester. You

will be identifying the errors that occur (and potential errors) in your experiment. It is not

acceptable to state that there were no errors; you will definitely lose points if you make

that statement.

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INTRODUCTION

CRISPR/Cas gene editing is a biotechnology tool that takes advantage of cellular

mechanisms that occur in living cells. This tool allows researchers to edit DNA by

creating mutations or inserting new DNA into a cell’s genome. The DNA that is modified

is the target DNA. A researcher may wish to modify a target gene so that it is silenced

(not expressed) or it is changed to express a different product. The uses of

CRISPR/Cas gene editing are growing rapidly, and this technology can be used to

modify both DNA and RNA in cells.

CRISPR/Cas gene editing is based on a naturally-occurring adaptive immune response

in Bacteria and Archaea. CRISPR refers to clustered regularly interspaced

palindromic repeats found in bacterial and archaeal genomes. These repeat

sequences are located near the Cas operon, which has multiple genes coding for

proteins that attack and degrade bacteriophage DNA. The Cas proteins are "CRISPR-

associated" proteins. While there are many types of CRISPR systems that exist in

bacterial and archaeal cells, the basics are outlined below, and are shown in Figure 1.

a. A cell containing the Cas operon has genes that produce multiple kinds of

proteins, including nucleases that cut DNA. Some of these Cas proteins can

bind to phage DNA that has entered the cell (Step 1 in Figure 1). Also at the

CRISPR locus are found palindromic repeat sequences near the Cas operon.

b. A Cas protein can recognize phage DNA by a three-nucleotide sequence on the

viral genome, called a protospacer adjacent motif (or PAM) site. If a Cas protein

recognizes a PAM site and binds to phage DNA, the Cas protein will cut out a

small section of the phage DNA.

c. The Cas proteins then integrate the small section of phage DNA into the bacterial

genome, as a "spacer" in between the palindromic repeats (Step 2 in Figure 1.)

d. The cell transcribes the section of DNA containing the spacer segments and

some other sections of the Cas locus, to produce what is known as the CRISPR

RNA, or crRNA (Step 3 in Figure 1). This crRNA is complementary to the piece

of viral DNA that was incorporated into the host’s genome. crRNA is combined

with another kind of RNA called trans-activating crRNA (or tracrRNA), to form

what is called guide RNA, or gRNA.

e. The gRNA binds to and is carried by a Cas endonuclease (often Cas9) through

the cytoplasm.

f. When new phage DNA enters the bacterial cell, if the phage DNA contains the

PAM site and is complementary to the gRNA, then the gRNA will bind to the

phage DNA, and the Cas protein will cut the phage DNA just upstream of the

PAM site. This destroys the phage DNA (Step 4 in Figure 1).

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Figure 1. Basics of the CRISPR system as it occurs in Bacteria and Archaea.

From: http://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/

Researchers have discovered that the Cas nucleases from bacteria will work in almost

any cell type to cut DNA. A crRNA and trcrRNA can be bioengineered to be

complementary to any target sequence of interest in a cell's genome, so it acts as a

guide RNA for where the Cas9 should cut the target DNA.

If a researcher combines a Cas endonuclease (such as Cas9) with a gRNA, and then

introduces them into a cell, the Cas nuclease will cut the DNA at the site that is

complementary to the gRNA at a site that is upstream of a PAM site. (It is imperative

that the researcher knows the PAM sites that the specific Cas nuclease being used will

recognize.) The method of introducing of the Cas nuclease and the gRNA can take on

many forms. In this series of labs, we will use plasmids to introduce Cas9 and the

gRNA in yeast cells.

The Cas nuclease typically makes a double-stranded break upstream from a PAM

site, which the cell will attempt to repair (see Figure 2 below). In a non-dividing cell,

repairs to double-stranded breaks may be done through non-homologous end joining

(NHEJ), which is error-prone and leads to mutations. If the targeted sequence, where

Cas nuclease cut, falls inside of a coding or regulatory sequence, then the mis-repaired

DNA can lead to silencing of the gene. Alternatively, if the cell is also provided with a

new section of template DNA, then the cell can (but will not always) repair the break by

incorporating the new template at the site where the Cas nuclease cut the DNA. The

integration of the new DNA is accomplished through a form of homology directed repair

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(HDR), such as homologous recombination. This leads to the insertion of new DNA

into the genome.

Figure 2. Recombination of DNA after double-strand cut by Cas nuclease.

From: https://www.cantechletter.com/2016/03/crispr-cas9-gene-editing-tool-may-lead-to-breakthrough-for-

cancer-autism/

Questions

1. What is the role of Cas9 in CRISPR in Bacteria?

2. Describe the structure and function of gRNA in CRISPR.

3. What is the role of the PAM site in CRISPR?

4. What happens to the DNA if there is a double-stranded break?

CRISPR/Cas GENE EDITING IN FISSION YEAST

The protocol you will use to modify S. pombe is adapted from Rodriguez-Lopez, et. al.

(2017). The basic series of steps (called the workflow) for this set of procedures is

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shown below in Figure 3. You should keep this workflow available during each lab, so

that you understand where you are in the experiment, what you are doing, and why.

Figure 3. Workflow for CRISPR/Cas gene editing in Schizosaccharomyces pombe. (A. Robinson, 2018)

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The details of the workflow are as follows: Step 1: Your instructor will assign two genes from S. pombe to each group. You will

identify their gene names, gene products, and phenotypic effects. You will then use a bioinformatics tool to design the guide RNA template (as DNA) for each gene. These sequences are sent to a biotechnology company that will construct the gRNA sequences (as DNA) for use in Step 2. These gRNA templates will be the gRNA genes used in the next steps.

Step 2: The gRNA genes (as DNA) are inserted into a bioengineered plasmid

(pMZ379) using polymerase chain reaction (PCR). This plasmid was specially developed to contain several features, including a gene for Cas9. This plasmid will be the vector by which the gRNA and Cas9 are introduced to the yeast cells. PCR will create many, many linear copies of the modified plasmid.

Step 3: A gel electrophoresis will be performed on the PCR products. This ensures

that the modified plasmid was actually copied many times.

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Step 4: The modified plasmid copies produced by PCR are linear. In order to be taken

up by the yeast, the plasmid must be circularized. This is done by inserting the plasmid into bacterial cells (Escherichia coli), which will make circular copies of the modified plasmid. This is done by transforming the E. coli with the modified plasmid.

Step 5: Once the modified plasmid is in circular form, it must be extracted from the

bacteria. The pure plasmid is recovered from the E. coli cells. Step 6: Once the modified plasmid is circularized, it must be checked to ensure that it

was truly modified (i.e., that the gRNA gene was actually inserted.) This is done by cutting the modified plasmid, as well as the unmodified plasmid, into fragments with restriction enzymes (i.e., doing a restriction enzyme digest). Once the two plasmids are cut into pieces, the sizes of the fragments can be compared. The modified plasmid should produce a larger fragment, which contains the gRNA gene, than the unmodified plasmid. The sizes of the fragments are compared by gel electrophoresis.

Step 7: The modified plasmid is then inserted into the S. pombe cells. This is done by

a chemical transformation of the yeast. The yeast will express the Cas9 gene and transcribe the gRNA gene from the plasmid, to make sgRNA. The sgRNA will guide the Cas9 enzyme to the target gene, and make a cut. The cell will attempt to repair the cut, and will likely cause errors, making the gene non- functional.

Step 8: The transformed yeast must be plated on growth media that will show if the

plasmid was taken up and the gRNA and Cas9 were expressed by the cells. If so, then the modified yeast should grow under different conditions from unmodified yeast. This step takes multiple periods of growth and plating.

MATERIALS • 1 internet-connected laptop

IDENTIFYING TARGET GENES AND DESIGNING sgRNA

In today’s lab, you will complete Step 1 of the workflow. Namely, you will identify the genes of interest, you will learn about those genes, and you will design gRNA sequences that are complementary to your target genes.

In order for target genes to be modified in yeast, the gRNA and Cas9 must be introduced into the cells. This will be done in Step 7 by the use of a plasmid (pMZ379) which has been specifically designed for use in S. pombe. pMZ379 was developed by Mikel Zaratiegui (Addgene plasmid # 74215; http://n2t.net/addgene:74215 ; RRID:Addgene_74215).

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The plasmid is shown in Figure 4 below. The unmodified plasmid contains several features of interest:

• a gene for Cas9

• an insertion site for the gRNA, next to a promoter

• an ampicillin resistance gene

• a nourseothricin resistance gene

• and many restriction enzyme cleavage sites.

Figure 4. pMZ379 restriction map. (From: https://www.addgene.org/74215/)

After the gRNA has been designed, the sequence of the gRNA will be sent to a biotechnology company, which will construct an actual sequence of DNA that matches the gRNA. These short sequences of DNA (also known as oligonucleotides, or “oligos” for short) will then be inserted into pMZ379. This creates a modified plasmid which can be inserted into the yeast cells. When the plasmid is introduced into the yeast cell, the genes on the plasmid will be expressed by the yeast’s enzymes. The Cas9 gene will be expressed, creating the Cas9 enzyme. The gRNA gene will be transcribed into a single-stranded RNA (sgRNA) that can attach to the Cas9, and guide it to the target gene. Remember, the Cas9

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enzyme will only attach at a target sequence where it can find a PAM site nearby. When the gene has been cut, the cell will attempt to repair the cut with NHEJ. This will likely cause errors in the repair, which will lead to a non-functional or silenced gene. The sgRNAs are fairly short sequences (~20 bp). Cas9 moves along the cell’s DNA, and will cut upstream from any place where there is a PAM site and a sequence that is mostly complementary to the gRNA. Since the complementary sequences are fairly short, it is possible for the Cas9 to find multiple sites at which to cut. This means that the Cas9 will sometimes cut at sites that are not the intended target, called “off-target” sites.

Questions

1. Describe the role of the plasmid in this protocol. (What is its purpose, what are its features that allow it to serve its purpose, how will it be used, etc?)

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Target Genes

1. Each group will be assigned two target genes to modify. Write your gene names in

Table 1 on page 13 of the handout.

2. Make sure that your laptop is connected to the internet.

3. Go to the Pombase database: https://www.pombase.org/

4. From the menu near the top of the page, choose Search, then Gene list. A new

window will appear with a box for you to enter the gene name. See Figure 5 below.

5. Once you have entered the gene name, click Lookup. The results of your query will

appear in a box near the bottom of the screen (Fig. 5).

Figure 5. Gene search window in Pombase.

6. Click on the number (e.g., “1”) in the results query box. A new window will appear

with the gene information.

7. Record the gene product in Table 1.

8. Record the gene’s location (chromosome number and number of nucleotides [nt

start to stop]) in Table 1.

9. Go to D2L and read the papers relating to your genes on the course website. Briefly

describe what silencing each gene will do to the phenotype of the cell in Table 1.

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Designing the sgRNAs

Designing the gRNA oligos by combing through the S. pombe genome yourselves would be exceptionally time-consuming. Thankfully, there is a database that will design the appropriate oligos for you!

This step uses the database CRISPR4P to design a guide RNA template. Once the name of the target gene to be cut is entered into the database, CRISPR4P will automatically generate a list of possible single-guide RNAs (sgRNAs) near PAM sites, along with primers. These primers are necessary, because in order to insert the oligo into the plasmid, the ends of the oligo sequence must overlap the sequences at the gRNA insertion site on the plasmid. Therefore, the oligos that you design must have the following features:

• They must be complementary to a section of the target gene that is near a PAM site.

• They must have ends that are complementary to the sequence at the gRNA insertion site on the plasmid.

1. Go to CRISPR4P (bahlerlab.infor/crispr4p).

2. Input the desired deletion target by gene name or coordinates on chromosome, as

shown in Figure 6 below. Click Submit.

Figure 6. Entering gene information into CRISPR4P.

3. Once the gene name is submitted, CRISPR4P will generate a list of suggested

sgRNAs (single-guide RNAs). The suggested sgRNAs are unique in the genome.

In the list, the numbers to the right show how many other sequences share the

column number of nucleotides. For example, Figure 7 below shows that, for the first

sgRNA in the list, there are eight sequences in the yeast genome that have eight or

more nucleotides also found in the sgRNA. For the third sgRNA in the list, there are

eleven sequences in the genome that have eight nucleotides in common with the

sgRNA. For the third sgRNA, there is only one sequence that has ten or more

nucleotides in common with the sgRNA. Ideally, you want to choose sgRNAs that

have very low numbers in the columns to the right, which indicate its uniqueness in

the yeast genome. This reduces the chances of the Cas9 enzyme cutting the

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genome at other sites, or creating off-target effects. Write the sequence of the first

sgRNA in the list in Table 1.

Figure 7. sgRNAs listed by uniqueness in genome. 4. Click on the circle to the left of the first sgRNA in the list. The suggested primers

and the PAM site information will appear below the box of sgRNAs, as shown in

Figure 8. Record the sequence of the PAM site in Table 1. There are two primers

(a forward and reverse) needed for each sgRNA. Choose the "Ligation-free

Primers" for the protocol used in this handout. Write the sequences of the

forward and reverse primers in Table 1.

Figure 8. Primers required for sgRNA.

5. Repeat Step 4 for the second sgRNA in the list.

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Gene 1: Gene 2:

Gene product

Location

What will silencing gene do to the

cell?

sgRNA 1 sequence

sgRNA 1 PAM

sgRNA 1 forward primer

sgRNA 1 reverse primer

sgRNA 2 sequence

sgRNA 2 PAM

sgRNA 2 forward primer

sgRNA 2 reverse primer

Table 1. Gene and sgRNA information.

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References

Rodríguez-López, M., C. Cotobal, O. Fernández-Sánchez, N. Borbarán Bravo, R. Oktriani, H. Abendroth, D. Uka, M. Hoti, J. Wang, M. Zaratiegui, and J. Bähler. 2017. A CRISPR/Cas9-based method and primer design tool for seamless genome edition in fission yeast. Wellcome Open Research 1:19. Version 2, May 2017