BIS7022-Genetic Editing with CRISPR/Cas9 – Medical Science Assignment Help

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In this workshop, we will explore how CRISPR/Cas9 can be used to generate precise genetic edits. Following a brief introductory tutorial, we will undertake a series of tasks to introduce basic concepts of genetic editing using online portals that greatly simplify this process. Since the first use of CRISPR for genetic editing almost 10 years ago, many new resources and technologies have been developed. The basic system has two main components: a guide RNA (gRNA, also termed crRNA, sgRNA), which includes 23 bp of RNA with complementarity to a genomic locus, and Cas9 protein (CRISPR associated protein 9), which plays a key role in the unwinding of DNA and cleaving DNA where complementarity exists between the gRNA and the target genomic locus.

Originally identified as repetitive sequences in bacteria that were later implicated as a defence mechanism against bacteriophages, it’s repurposing for genetic editing, principally through the work for Emmanuelle Charpentier, Jennifer Doudna (recipients of the 2020 Nobel Prize for Medicine) and Fang Zhang, has led to an explosive use across biology and medicine. The simplicity of the system compared with other genetic editing tools – such as TALENs – led Rudolf Jaenisch (who made the first transgenic mouse in 1974, and reported the first CRISPR’d-organism in 2013) to notably quip that “any idiot can do it”. While the system is well-geared for experimental research, it is generally unsuitable for medical purposes due to off-target effects and chromosomal errors (see Alanis et al, 2021 in the extended reading). In this workshop, you will be introduced to a basic outline of genetic editing, and use simple online portals for the design and validation of a gene targeting strategy.
                    

Part 1: Designing gRNAs to your gene of interest

We will first use a bioinformatic platform operated by a commercial company, Synthego. There are many similar programs; today we’re using Synthego’s platform as it’s very straight forward and accessible. The goal of the first exercise is to design gRNAs directed to a target gene to produce a loss-of-function allele.

1. The first step is to choose a gene of interest you wish to edit. This can be any gene that you’re currently working with (or planning to do so). Alternatively, chose a gene that you’re aware of from a disease state (eg, TP53 and cancer, or Erythropoietin – EPO – in red blood cell production).

2. List your gene of interest and organism.

3. Select your genome (the species you plan to target – eg. choose Homo Sapiens) and then type in your gene name. Then click “search”.

4. The algorithm will usually produce four preferred (“top-ranked”) guide RNAs based on four criteria. These are:
a. Early coding region (located near the start codon)
b. Common exon (exon present in most isoforms)
c. High activity
d.mMinimal off-targets (for example, scores of “0,0,0,2,5,36” denotes no genomic locus with 0, 1 or 2 mismatches, 2 loci with 3 mismatches, 5 loci with 4 mismatches etc).

Q2) This algorithm is best suited to creating loss-of-function alleles. Briefly describe why each criteria is an important consideration when choosing an appropriate target sequence.

5. At the top of the web page are two tabs (see picture below); “recommended guides”, and “all guides”. Move across to the “all guides” tab, which brings to you additional targeting sites. Depending on your gene, you may then be able to navigate among each exon (this may be useful if planning to edit a specific exon).

Q3) For your two top ranked gRNAs, note the chromosome, cut site, exon, on-target score and off targets in the table. 

Q4) Why is the guide in rank 1 preferable to that of rank 2? (ie, a better target score? Less off targets? Earlier exon? Etc) Why is this important?

6. Returning to the web browser, click on the top-ranked gRNA sequence (green text). This opens a new page that gives you the gRNA alignment with the genome, showing you the PAM site (blue text) and location of the cut site (orange triangles). 

Q5) Will your gRNA (green text) bind to the positive strand (upper), or the negative strand (lower) of your DNA?

7. Returning to the web browser: by scrolling down the page, you’ll find a list of the predicted off-target sites, showing “mis-matches”, their genomic location, and whether they occur in a protein coding gene.

Q6) Why would an off-target site within a gene be an issue for your design?

8. Note which chromosome your gene of interest is located on (see table you completed in Q3). Click on the right of the page, click “Filters”, and then in the centre of the page below the “Chromosome” text box, type your chromosome (eg, “Chr17”).

Q7) Are there any predicted off-target sites on the same chromosome? And if so, what distance are they (in nucleotides) from your gene of interest?

Q8) Why could an off-target site linked (ie, closely located) to your gene of interest create an issue?

9. Return to the previous webpage (from step 3). To create a deletion, it’s common to use two gRNAs closely located to one another; when each genetic locus cut, the intervening sequence may be removed and the two ends repaired through non-homologous end joining (NHEJ). Using two gRNAs placed >50bp apart can often generate a deletion of the intervening sequence. Make a note of two gRNAs that are located >50 bp apart, and complete the table below
In a practical sense, generating these gRNAs and delivering them to your cell of interest with the appropriate Cas9 enzyme are all that are required for creating a genetic edit. The next challenge is to identify clones (all arising from a single edited cell) that carry your genetic edit. In part 2, you will design a simple PCR-based strategy to identify clones carrying the genetic deletion you designed above.

Part 2: Detecting genomic deletions by PCR

In this section, you will design a genotyping strategy to identify cells with your desired genetic edit. In part 1, you have designed your gRNAs; the next experimental step is to get this gRNA into your cell (via transfection, injection, viral transduction etc.) together with Cas9 (either via plasmid, RNA, or protein, depending on the context; not these steps are not covered in this workshop). Upon getting each component into your cell or organism of interest, the third step is to identify which of your cells carry desired genetic edits.

The easiest approach is to examine the genetic sequence through a PCR-based strategy. Polymerase chain reaction (PCR) works by the annealing of two oligonucleotide sequences (primers) to genomic DNA, and amplifying the intervening sequence using a polymerase called Taq. (If you need a reminder how PCR works, see the first 3 minutes of this video: https://youtu.be/matsiHSuoOw). If there’s a large deletion in the genomic DNA, you can visualise this by gel electrophoresis as a shorter PCR (DNA) product. One common approach to identify cells/clones/organisms carrying a deletion is to amplify the genomic locus by PCR and screen a large number of these clones and identify those carrying a deletion. In part 2, we will apply your primer designing skills for this purpose.

1. First, obtain the sequence surrounding your gene of interest. If you’ve selected a human gene, one way to find this sequence is to go directly to the genome: GRCh38.p14 – hg38 – Genome – Assembly – NCBI (nih.gov)
If your DNA is of another species, you can easily search for this by following the same link and then typing your species in the search box at the top of the page (eg. “mouse”, and then select the genome, eg “GRCm39”).

2. Within the reference genome, each chromosome is presented as a unique Genbank or RefSeq sequence. Scroll down to the GenBank sequence, as shown in the image below. Click on either the GenBank sequence or RefSeq sequence for the chromosome where your gene is located. For example, for a gene located on Chr5, select “NC_000005.10”

3. The next step is to extract your DNA of interest. In the new tab that opens, the most simple way to do this is to select “Change region shown” to specify the DNA sequence surrounding your two gRNA target sites from part 1. Add and subtract 200-300 bp either side of your sequence. Eg. for a single gRNA located at 55,779,937 insert the following: 55,779,737 to 55,780,137, and click “update view.” (Note, you will need to remove the commas.)

Q9) Note here the genomic regions you selected:

4. On the updated page, click “FASTA” to retrieve your sequence. Eg;

Q10) Paste your FASTA sequence here: (FASTA is a format for DNA sequences, “fast-all”, where the sequence is presented with single letter codes without intervening sequences. The FASTA format also includes a description line beginning with “>” which gives a name or unique identifier to your sequence; it’s not necessary to include this first line here).

5. Now that you have retrieved a genomic sequence, you will next design primers to amplify this sequence (which includes the gRNA target sites you identified above) from genomic DNA. A very simple tool for this purpose is to use “Primer-BLAST”, which you can find here: https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi This tool generates candidate primer pairs (using “Primer3”), and then checks the target specificity of these primer pairs by performing a sequence alignment to the remainder of the genome (BLAST, as you learned about in a previous workshop). The effect of combining these two tools together in one step is to produce specific primers that are unique to your genomic locus of interest.

6. Copy your >400 bp FASTA sequence and paste into the Primer-BLAST search tool. There are several parameters that can be modified that are specific to your query;

a. Under “Primer parameters” the default is to produce amplicons (distance between primer pairs) of 70-1000bp. Change the minimum to “150”. The reason for this is to create an amplicon of sufficient size that covers both your gRNA targeting sites, and with sufficient size to detect your potential deletion.

b. Under “Primer pair specificity checking parameters” ensure that “specificity check” is selected. Make sure the organism is the same as the one you’ve designed a gRNA to. Eg. if you’re working with humans, type “Homo sapiens”.

c. Select “show results in new window” then click “get primers”. This will open a new window, and may take several minutes to complete the design of primers and then BLAST the results.

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