Capstone Activity
Identification and Bioinformatics Analysis of Cas9 Target Sites
The ability of the CRISPR-Cas9 system to accurately and permanently edit genomes has major
implications for the treatment of diseases. Some diseases, such as coronary artery disease, sickle celldisease, and cystic fibrosis, are caused by genetic mutations. A CRISPR-based therapy that can edit
the genomic DNA in cells may be able to correct those mutations.
Though this type of therapy is promising, it is not as clear-cut as it may seem on the surface. As withany therapy, there are risks involved that must be analyzed and understood before testing in humans.
For example, off-target effects, where a gene or DNA sequence other than the intended target is edited,
can have dire effects on an organism. These types of risk can never be completely eliminated, but theirprobabilities and the conditions in which they may occur must be evaluated.
One of the first steps in designing a CRISPR-based therapy is identifying a gene-editing strategy andselecting a Cas9 target site to cut. In this activity, you will research the genetic basis for a disease andexplore a CRISPR-based gene-editing strategy: replacing, inserting, or deleting a sequence. Then you
will identify potential Cas9 target sites and use the basic local alignment search tool (BLAST) from the
National Center for Bioinformatics Information (NCBI, part of the National Institutes of Health, NIH) to
search for similar sequences in the human genome. Using these data, you will analyze your potential
Cas9 target sites for risk of off-target effects to identify the most promising candidate for a CRISPRbased therapy.

Part 1. Identify and Catalog Target Sequences
1. Read the background information about the disease you are investigating.
		 Discuss with your group and answer the disease-specific reading questions.
2. Scan the provided DNA sequence and identify all potential Cas9 target sequences.
		 Consider the following:		 • A Cas9 target site includes a 20-nucleotide protospacer sequence followed downstream by			 an appropriate PAM sequence (5'-NGG) in the 5' to 3' direction. Therefore, a target sequence			 is 23 nucleotides long		 • A search for PAM sequences first may speed up the process		 • Target sequences can be found on either DNA strand, but always in the 5' to 3' direction
3. Select 2–4 candidate target sequences to investigate.
		 Record each sequence in the table below, using the following naming convention: Gene name		 abbreviation-your initials-#. For example, GENE9-TRP-1.
Target Sequence Position # of First
Name
Nucleotide

22

Position # of Last
Nucleotide

23-Nucleotide Target Sequence, 5' to 3'

Part 2. Perform BLAST Search for Off-Target Sequences
A complete or partial Cas9 target sequence can sometimes be found elsewhere in the human genome,
so an sgRNA designed against such a site may guide Cas9 to cut off-target sites. You will use thebioinformatics software BLAST to find genes with sequences that completely or partially match thetarget sites you selected above.
1. Prepare your results table.
		 Use the bioinformatics results table provided in the student guide or recreate it in a spreadsheet
		program.
2. Perform a BLAST search.
		 The BLAST interface changes frequently. The following instructions and screenshots may deviate
		 slightly from your experience on the site.
		2.1. Visit blast.ncbi.nlm.nih.gov
		2.2. Click Nucleotide BLAST. Copy your target sequence from your table and paste it					under Enter Query Sequence.

		2.3. Under Choose Search Set > Database, select Human RefSeqGene sequences					(RefSeq_Gene).

explorer.bio-rad.com

23		2.4. Select Show results in a new window and click BLAST.

		 2.5. The BLAST search may take a few minutes to complete, depending on the server					 usage volume. When complete, a screen similar to that below appears.

3. Review the BLAST search results.
		 3.1. One of the top results should be an exact match for your query in the gene you are					 working with. Click the description link and review the information provided. Then					 return to the BLAST results page.
		 3.2. Click other links in your results list to orient yourself to the information provided.
					 Then return to the BLAST results page.

24		 3.3. Select all the sequences (select all), click to open the Alignments tab, then select					Alignment view > Query-anchored with letters for identities.

		 3.4. Review the sequence information.

					 a. Your sequence (Query) appears at the top of the results for easy reference. The
							 remaining rows display nucleotide sequences from genes that match or partially							match your query sequence.
					 b. The links at left are the accession numbers for reference genes. Each gene in the							database has its own accession number.
					 c. To the right of each accession number is the start position of the alignment							 sequence, followed by the sequence and the number of the nucleotide at the							 end position. Most alignment sequences will not match the full length of the							query sequence.
		 3.5. One of the top results should be an exact match for your query and be located in the					 gene you are working with. Click the accession number. Ensure the information given					 for this match is correct (that it matches the gene you are working with). If it does not					 match exactly, check that your query sequence is correct.

explorer.bio-rad.com

25

4. Annotate your results.
		 4.1. In your bioinformatics results table record five alignment results (or as many as					 possible if there are fewer than five) whose sequences include the PAM sequence					5I-NGG aligned with that of the query sequence, where N is any nucleotide. Use the					 accession number link to retrieve additional information to fill in the table. There may					 be multiple results for a single accession number.
		 4.2. Highlight any exact alignment matches in genes other than your intended target.
		 4.3. Circle the longest alignment sequence(s) other than your intended target.
5. Repeat steps 2–4 for each of your target sequences.

Focus questions
A. Why might there be multiple results from a single accession number?

B. How might an exact alignment match be an off-target cut site?

C. Does the presence of alignment matches indicate higher or lower risk of off-target effects?

26

Part 3. Evaluate Candidate Target Sequences
Because the sgRNA sequence determines where Cas9 cuts DNA, it may cut DNA anywhere it encounters
a complementary sequence, even in undesired locations. Such unintended cuts are called off-targets.
Experiments have shown that Cas9 sometimes cuts DNA even when there are mismatches between
the sgRNA and DNA sequences. This means DNA sequences that only partially match the sgRNA havethe potential to be off-target cut sites, although the PAM sequence is still required. In general, the moremismatches there are between a DNA sequence and the sgRNA, the less likely Cas9 is to cut.
1. Develop three criteria that could be used to rank the candidate target sites according to their		safety for therapeutic use. Use the alignment results you have collected in your BLAST search
		 as well as the information you considered from the readings and focus questions as you		 select criteria. For each criterion, explain how it will help you evaluate a potential target site.

2. Use your criteria to evaluate and rank the candidate target sites you selected in Part 1,
		 step 3. Write a claim about which candidate target site is the best option for your therapeutic
		 application, based on your evaluation, and provide reasoning.

explorer.bio-rad.com

27

Focus questions
A. If you were to continue evaluating the candidate target sites for use in a therapy, what are		 two additional pieces of information or experiments that would help you?

B. What health problems could arise from off-target CRISPR-Cas9 activity?

C. How would you decide whether the risk of off-target activity for a CRISPR-Cas9 therapy		 is low enough to be considered safe?

D. Should off-target effects be considered for nontherapeutic CRISPR experiments in the		 laboratory? Explain why or why not.

E. Do you think there should be differences between how off-target risk is evaluated for		 CRISPR-based therapies and for laboratory CRISPR experiments?

28

Coronary Artery Disease
Background
Cardiovascular disease is the leading cause of death worldwide, claiming over 17 million lives annually.
One type of cardiovascular disease, coronary artery disease (CAD), in which blood vessels near theheart become narrowed due to plaque buildup, claims nearly 8 million lives each year. Lowering levelsof low density lipoprotein (LDL) cholesterol has been shown to effectively reduce risk of CAD. LDL
receptors (LDLR) in the liver clear LDL from blood plasma. However, levels of LDLR are themselves
reduced by proprotein convertase subtilisin/kexin type 9 (PCSK9), a serine protease that binds anddegrades the receptors (Figure 6). People with mutations in the PCSK9 gene commonly have lower
levels of LDL cholesterol likely because they have higher levels of the LDL-clearing receptors.

Gene-Editing Therapy Strategy
A goal of gene-editing therapy may be to reduce or eliminate PCSK9 enzyme function. One strategy is
to disrupt the gene by making a cut within exon 1 and allowing nonhomologous end joining (NHEJ) to
occur. This strategy would reduce levels of functional PCSK9 enzyme in the liver, which would reduce
degradation of the LDL receptors to allow more removal of LDL cholesterol from the bloodstream.
PCSK9 Present
PCSK9

PCSK9 Absent
LDL

LDL

LDL–LDLR–PCSK9Complex formation

LDL–LDLR
Complex formation

LDLR

Complexendocytosis

Complexendocytosis

LDL
Degradation

LDLR
Recycling

Degradationof LDL, LDLR,
and PCSK9

Fig. 6. Degradation of LDL in the presence and absence of PCSK9.

explorer.bio-rad.com

29

Focus questions
A. Describe how disrupting PCSK9 would impact gene expression. Draw a model that
		 illustrates your description.

B. Describe two potential advantages and two potential disadvantages of administering		 such a therapy to liver cells only.

C. Is this gene editing strategy an example of replacing, inserting, or deleting a sequence?

PCSK9 gene sequence information
Gene Accession Number:Gene Reference:
Gene Abbreviation:
5053

NG_009061.1
Homo sapiens proprotein convertase subtilisin/kexin type 9
PCSK9 *

5'-GGTGCATCTGACTCCTGTGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGT-3'3'-CCACGTAGACTGAGGACACCTCTTCAGACGGCAATGACGGGACACCCCGTTCCA-5'

5106

The sequence below is an excerpt of PCSK9 exon 1 nucleotide position 5,387 to 5,446.

5387 5'-GGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCGAGGAGGACGGCCT-3' 5446
3'-CCTGCTCCTGCCGCTGATGCTCCTCGACCACGATCGGAACGCAAGGCACCTCCTGCCGGA-5'

98756
30

***
5'-TTCTGTTCTCAGTTTTCCTGGATTATGCCTGGCACCATTAAAGAAAATATCATCTTTGGT-3'3'-AAGACAAGAGTCAAAAGGACCTAATACGGACCGTGGTAATTTCTTTTATAGTAGAAACCA-5'

98815

Sickle Cell Disease
BackgroundSickle cell disease is an inherited blood disorder in which a person’s red blood cells become sickleshaped, which increases the risk of blood clots. When we scrape a knee or cut a finger, blood clots
form externally to create a scab over the wound and promote healing. When blood clots form internally,
however, they can block blood vessels and cause pain or even death. Approximately 100,000 people
die of complications from sickle cell disease each year.
Sickle cell disease is caused by a single nucleotide polymorphism (SNP) in the hemoglobin B (HBB)
gene called rs334. People homozygous for adenine at rs334 produce normal hemoglobin while thosehomozygous for thymine at rs334 produce sickling hemoglobin and have the disease. Heterozygous
individuals do not exhibit symptoms of the disease and have increased resistance to malaria, which
increases their evolutionary fitness in regions where malaria is common.

Gene-Editing Therapy Strategy
The goal of gene-editing therapy for sickle cell disease is to allow expression of functional non-sickling
hemoglobin. Red blood cells, which carry hemoglobin, are formed from hematopoietic stem cells inbone marrow (Figure 5). A potential gene-therapy strategy is to harvest hematopoietic stem cells froma patient, edit the HBB gene in those cells, and reintroduce them to the patient. Using a patient’s own
cells greatly reduces the chance of rejection by the patient’s immune system. CRISPR-based gene
editing is used to replace thymine with adenine at rs334 by making a cut near the SNP and introducing
the correct sequence using homology-directed recombination (HDR).

CommonLymphoid Progenitor
Bone

Hematopoietic
stem cell

Multipotent
Hematopoietic
stem cell

CommonMyeloid Progenitor

Erythrocytes(red blood cells)

Red marrow tissue

Fig. 5. Differentiation of hematopoietic stem cells into red blood cells.

explorer.bio-rad.com

31

Focus questions
A. For this gene-editing strategy, why is it useful to edit the DNA of only hematopoietic
		stem cells?

B. What other cells could be edited to achieve similar results? List two potential advantages
		 and two potential disadvantages of editing these cells instead of hematopoietic cells.

C. Is this gene editing strategy an example of replacing, inserting, or deleting a sequence?

HBB gene sequence information
Gene Accession number:Gene Reference:								Gene Abbreviation:

NG_059281.1
Homo sapiens hemoglobin subunit beta (HBB),
RefSeqGene on chromosome 11
HBB

The sequence below is an excerpt of HBB, nucleotide position 5,053 to 5,106, with rs334 shown
bolded and with an asterisk.
5053

32

*
5'-GGTGCATCTGACTCCTGTGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGT-3'3'-CCACGTAGACTGAGGACACCTCTTCAGACGGCAATGACGGGACACCCCGTTCCA-5'

5106

5387 5'-GGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCGAGGAGGACGGCCT-3' 5446
3'-CCTGCTCCTGCCGCTGATGCTCCTCGACCACGATCGGAACGCAAGGCACCTCCTGCCGGA-5'***

Cystic Fibrosis
Background
Cystic fibrosis (CF) is an autosomal recessive disease that affects the lungs, pancreas, and small
intestine. The disease affects about 70,000 individuals worldwide. It causes buildup of viscous mucus
in these organs and frequently leads to severe lung infections. If untreated, most CF patients do not live
past their 20s. The disease is caused by mutations in the cystic fibrosis transmembrane conductanceregulator (CFTR) gene on chromosome 7, the most common of which is an in-frame deletion of threebase pairs in exon 11 that codes for phenylalanine (F508del). CFTR is a protein that transports chloride
ions across cell membranes, which is critical to effective clearing of mucus from airways (Figure 7). The
F508del mutation impairs the normal production of CFTR.

Gene-Editing Therapy Strategy
The goal of cystic fibrosis gene-editing therapy is to correct the CFTR gene in lung epithelial stem
cells. A therapeutic drug will likely be given by inhalation to target the lungs and the edit will occur invivo. CRISPR technology would be used to create a cut in exon 11 in the vicinity of the CFTR F508del
mutation and use CRISPR-mediated homologous recombination to replace the mutation with a healthy
version of the gene.
Normal Patient
Normalmucus layer

Normal CFTR
Epithelial cell
membrane
Normal lungs

Normallung airway

Chloride ionsPatient with CF
Thickmucus layer
No CFTR
Epithelial cell
membraneLungs with CF

Lung airway
with CF

Chloride ionsFig. 7. The mucus layers in lung airways of healthy patients and those with CF.

explorer.bio-rad.com

33

Focus questions
A. Describe how editing CFTR would impact gene expression. Draw a model that illustrates
		your description.

B. What are two potential advantages and two potential disadvantages of administering a		 cystic fibrosis gene editing therapy by inhalation instead of orally, by injection, or		another method?

C. Is this gene editing strategy an example of replacing, inserting, or deleting a sequence?

CFTR gene sequence information *5053 5'-GGTGCATCTGACTCCTGTGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGT-3'
5106
Gene Accession
Number: NG_016465.4
3'-CCACGTAGACTGAGGACACCTCTTCAGACGGCAATGACGGGACACCCCGTTCCA-5'
Gene Reference:
Homo sapiens CF transmembrane conductance regulator (CFTR)
Gene Abbreviation:
CFTR
5387 5'-GGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCGAGGAGGACGGCCT-3' 5446

3'-CCTGCTCCTGCCGCTGATGCTCCTCGACCACGATCGGAACGCAAGGCACCTCCTGCCGGA-5'
The sequence
below is an excerpt from exon 11 of CFTR, nucleotide position 98,756 to 98,815, withthe three-nucleotide mutation shown bolded and with asterisks.
98756

34

***
5'-TTCTGTTCTCAGTTTTCCTGGATTATGCCTGGCACCATTAAAGAAAATATCATCTTTGGT-3'3'-AAGACAAGAGTCAAAAGGACCTAATACGGACCGTGGTAATTTCTTTTATAGTAGAAACCA-5'

98815

TargetSequence
Name

Target Sequence(5I to 3I)

Gene
Accession
Numberof Result

Gene Name
Alignment Sequence(Abbreviation) (5I to 3I)

Start
Nucleotide#

End
Nucleotide#

LengthAlignment
Number ofSequence
Mismatches
IncludesCorrect PAM?
(Y/N)

Bioinformatics table

explorer.bio-rad.com

35