8: CRISPR
Summary
CRISPR/Cas9 is a gene editing technique that allows scientists to “cut and paste” genes resulting in a gene “knockout” or insertion of new DNA or specific sequence changes, depending on how the technology is used.
Also known as:
CRISPR/Cas9, clustered regularly interspaced short palindromic repeats
Samples needed
Cells to be modified, guide RNA to lead Cas9 to DNA sequence of interest
Method
The CRISPR/Cas9 genome modification system was discovered within bacteria, which use CRISPR as a defense against previously encountered viruses. The system involves a guide RNA (gRNA) which is complementary to part of the gene of interest, and a nuclease, most commonly the Cas9 enzyme. Cas9 unzips the DNA and allows the RNA to bind to it, then Cas9 cuts both strands of DNA.
CRISPR/Cas9 can be used to achieve multiple different outcomes. When Cas9 creates a double-strand break, the cell will attempt to repair the sequence with normal cellular repair processes, namely homology-directed repair (HDR) or non-homologous end joining (NHEJ). When NHEJ is used, random small insertions and deletions occur, often resulting in a functional knock-out, i.e. the gene of interest no longer produces a functional product. This is the most common outcome if the researcher does not provide a plasmid containing a repair template. However, if the researcher provides a DNA sequence containing homology arms flanking the sequence of interest, the cell can use HDR to repair the break, resulting in a change to or insertion into the genome. This can result in a gene knock-in.
A plethora of CRISPR variants exist. One of these is base editing. In this application, either cytidine deaminase or adenosine deaminase is fused to a Cas9 nickase, resulting in either cytidine-to-uridine or adenosine-to-inosine changes. The ability of guide RNAs to target proteins to a particular location in the genome can also be used for applications other than genome editing. For instance, if a catalytically dead Cas9 is fused to a transcriptional activator or repressor, the CRISPR system can be used to control gene expression. GFP-dCas9 can be used to track the location of a particular DNA sequence.
Controls
The most important control should show scientists that the DNA has been modified in the correct place. This can involve DNA sequencing, or if the gene is being disrupted, use of a western blot to show loss of target protein expression.
Interpretation
The TRAC locus contains the gene for the constant region of T-cell receptor α (TCRα). In Eyquem et al., the authors set out to disrupt the TRAC locus and instead insert DNA encoding a chimeric antigen receptor (CAR), to create therapeutic CAR T cells for leukemia treatment. If the genomic modification was successful, the T cells should no longer express the T-cell receptor, and instead express the chimeric antigen receptor. Panel b shows the results of a flow cytometry experiment. When reading the flow results, each dot is one cell; the height on the y axis shows how much TCR is present on the cell’s surface, whereas the distance along the x axis shows how much CAR is present. In the first graph, cells were supplied with the repair template containing the CAR gene, but no gRNA or Cas9. Essentially all cells show high levels of TCR and low CAR, as expected. In the second, cells were supplied with gRNA and Cas9, but no repair template. In this case, one would expect small insertions or deletions in the TRAC locus through the use of NHEJ. As expected, some cells in this group show much lower TCR levels, indicating that a functional knockout has been produced. In the final graph, all CRISPR components plus the repair template were supplied. In this case, there is a population of cells that show low TCR levels and high CAR levels, indicating that the locus has been successfully modified with the CAR DNA cassette.
Image Descriptions
Figure 1 image description:
Panel a: A schematic of a genomic locus. The top line is entitled “rearranged TCRα,” with TRAV and TRAJ exons upstream of the TRAC locus. The gRNA targets exon 1 of the locus, while the STOP codon is found in exon 3. The second line is entitled “AAV” for the adeno-associated virus that contains the CAR gene cassette. It shows a left homology arm (LHA), the CAR cassette (SA-2A-1928z-pa-1), then right homology arm (RHA). The diagram shows that the LHA corresponds to the sequence upstream of exon 1 in the TRAC locus, and the RNA begins toward the 3’ end of exon 1.
Panel b: Density dot plots, the result of flow cytometry experiments. Percent of cells in each region shown in table below.
|
Cas9/gRNA - AAV MOI 1 x 106 |
Cas9/gRNA + AAV MOI 0 |
Cas9/gRNA + AAV MOI 1 x 106 |
|||
|---|---|---|---|---|---|
|
Percent of cells |
TCR high, CAR low (upper left) |
96.9 |
29.9 |
20.9 |
|
|
TCR low, CAR low (lower left) |
2.97 |
69.9 |
30.7 |
||
|
TCR low, CAR high (lower right) |
0.029 |
0.14 |
45.6 |
||
|
TCR high, CAR high (upper right) |
0.13 |
0.049 |
2.82 |
Thumbnail
"GRNA-Cas9.png" ↗ by marius walter is licensed under CC BY-SA 4.0 ↗.
Description: CRISPR Cas9 system
Authors
Original author: Anne E. Ebersold, Roger Williams University, April 25, 2022. Reviewed by Katherine Mattaini.
Later expanded & edited by Katherine Mattaini, Tufts University, August 21, 2024.
- Eyquem, J., J. Mansilla-Soto, T. Giavridis, S. J. C. van der Stegen, M. Hamieh, K. M. Cunanan, A. Odak, M. Gönen, and M. Sadelain. 2017. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543:113–117. ↵
General sources:
Medline Plus ↗ [medlineplus.gov]
"How CRISPR works" ↗ [www.sciencenewsforstudents.org]