CRISPR/Cas9 Genome Editing

What is CRISPR/CAS9?

CRISPR-Cas9 originates from bacteria as a mechanism to fight viral invaders from taking over. As a crucial part of the immune system, it is responsible for destroying the genome of any invaders by binding and cutting specific DNA sequences. This system provides acquired immunity for the bacterial cell but also has potential for genome editing in the future. CRISPR stands for “clustered regulatory inter-spaced short palindromic repeats”. This refers to the organization of the partially palindromic repeated DNA sequences found in bacteria. Cas9 is a protein associated with CRISPR that is used to recognize and cut out foreign DNA with the help of guide RNAs. Guide RNAs have a specific sequence that is complementary to the invading viral DNA. It will then attach to this viral DNA allowing the Cas9 protein to cut at a specific location. This system targets specific sequences in the genome and is able to edit the DNA at that location. Scientists have been using CRISPR-Cas9 as a tool to modify genes in organisms and to fix mutations that could possibly be fatal.⁵

How does it work?

There are two molecules that help create a mutation in the DNA. These molecules are the Cas9 protein and the guide RNA which is also known as gRNA. The gRNA has a set of bases that are able to target a specific sequence because they are complementary to that sequence. Once the gRNA finds this sequence, it will attach allowing the Cas9 protein to cut both strands. The cell will then try to repair the damaged DNA. Because the repair system is not perfect, a few bases will be left out causing a mutation. This mutation will cause the gene to function improperly or not at all.⁵

This shows the how CRISPR/Cas9 works
Photo Credit: Genome Research Limited

Bacteria have sequences in their DNA, that came from an attack by viral DNA, known as “spacers” that act as a genetic memory. Just like our immune system, these spacers remember previous infections and will recognize that infection again. If a recognized virus decides to invade a bacterial cell, the CRISPR system will cut up the DNA sequence that matches with the spacer sequence.

There are 3 steps of CRISPR

1.  Adaptation
2. Production of CRISPR RNA
3. Targeting

Adaptation happens when invading viral DNA is inserted as new spacers. This is important for the cell’s genetic memory to help recognize the virus again. Step 2 involves the production of CRISPR RNA. Repeats and spacers go through transcription that result in a single chain of RNA. The RNA produced is cut into pieces known as CRISPR RNA. This then needs to be able to guide the immune system to the sequence they are matched with allowing the foreign sequence to be cut and destroyed.¹

Future of CRISPR/CAS9

A lot of the research is still using animals as models or human cells that have been isolated to prevent any mistakes. This unique targeting system has potential for use in gene therapy to help fix mutated genes in humans. Scientists are eager to find a way to make CRISPR-Cas9 bind and cut accurately. There are two ways that this can be done:

• Design more specific gRNAs by using information about the DNA sequence and off target behavior of different versions of the complex
• Have a Cas9 enzyme that will cut a single strand of target DNA and not a double strand. This will reduce the chance of the Cas9 cutting in the wrong place.⁵

Latest research in CRISPR/Cas9 genome editing

A study done by Itamer Harel, Dario Riccardo Valenzano, and Anne Brunet provides strategies for genome engineering in the African turquoise killifish.  This particular vertebrate was chosen because of its short life span which is between 4 and 6 months. The protocol being used takes advantage of this short generation time and creates stable lines in about 2 to 3 months. ” This protocol provides powerful genetic tools for studying vertebrate aging and aging-related diseases.”²

This second study is written by  Dacheng Ma, Shuguang Peng, and Zhen Xie. This study looks at the integration and exchange of split dCas9 domains for transcriptional control in mammalian cells. Their discussion starts off by saying that the split Cas9 system was shown to be delivered in vivo by using recombinant adenovirus-associate viruses. The design the authors provided contain a method that can be used to reduce the size of synthetic circuits. This can be done by intergrating and switching Cas9/dCas9 domains that were fused with different functional domains. “Such a CRISPR-Cas9 system will be particularly appealing in biomedical applications in which viral delivery vehicles with a restrictive cargo size are preferred.”³

This final study researches double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.  The authors included four main points that their article covers.⁴ This includes;

• Cas9 nickase can facilitate targeted DNA double-strand break using two guide RNAs
• Double nicking of DNA reduces off target mutagenesis by 50- to 1,000-fold
• Multiplex nicking stimulates homology directed repair, microdeletion, and insertion
• Double nicking provides efficient modification of mouse zygotes

References

1.  CRISPR: A game-changing genetic engineering technique. Science in the News. 2014.

2. Harel I, Valenzano DR, Brunet A. Efficient genome engineering approaches for the short-lived African turquoise killifish. Nature Protocols. 2016;11:2010–2028.Ma D, Peng S, Xie Z. Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells. Nature Communications. 2016 Oct 3.

3. Ma, D. et al. Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells. Nat. Commun. 7, 13056 doi: 10.1038/ncomms13056 (2016).

4.  Ran AF, Hsu PD, Lin C-L, Gootenberg JS, Trevino AE, Konermann S, Scott DA, Inoue A, Matoba S, Zhang Y, et al. Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. 2013;155(2):479–480.

5. What is CRISPR-Cas9? yourgenome.org. 2016 Jun 7 [accessed 2016 Nov 3]. http://www.yourgenome.org/facts/what-is-crispr-cas9