ik heb er op de uni een stuk over geschreven. wellicht dat jullie hier iets meer aan hebben, je kunt uiteraard dit ook vragen:
The genome is edited by the type II CRISPR/Cas9 technique, which is, contradictory to other genomic alterations, suitable for various species and not leaving “scar” sequences. This CRISPR-associated protein 9 nuclease (Cas9) system is derived from the bacterial strain Streptococcus pyogenes. In contrast to previous techniques like Zinc finger nuclease and transcription activator-like effector nucleases which use DNA-protein interactions, this technique is RNA-guided. In the CRISPR/Cas9 technique, Cas9 promotes a DNA double-stranded break (DSB) at a specific target sequence. A nuclease-induced DSB can be repaired by either nonhomologous end-joining (NHEJ) or homology-directed repair (HDR). NHEJ can result in various insertions or deletions of varying lengths, and will thus result in unpredictable outcomes. This is because degradation of a few base pairs (around 10) will take place at the each side of the double stranded DNA. After this, ligase will stick the two double stranded DNA parts back together, however, a few base pairs will be missing. Therefore, it could potentially disrupt the translational reading frame or the binding sites of promoters and enhancers. Using HDR, the other natural repair mechanism, the genome can be modified in a highly specific and efficient way. Since the double stranded DNA is modified back to single stranded DNA, no base pairs will become missing. HDR introduces desired mutations with the use of exogenously supplied DNA donor templates (Sander J.D. et al., 2014).
Originally, the CRISPR/Cas9 system is a defence mechanism from bacteria against foreign nucleic acids. Transcripts from the CRISPR gene of the bacteria are transcribed into CRISPR
11/30
RNAs (crRNAs) which are complementary sequence to the invading genome. This complementary sequence is called the protospacer. This crRNA forms a complex with the transactivating CRISPR RNA (tracrRNA) and eventually form a complex with Cas9 nuclease. The protospacer binds specifically and thereby ensures that Cas9 cuts at the right place, see Figure 8. To make this complex unable to target its own genome where the protospacer coding DNA is present, a special DNA sequence, called protospacer adjacent motifs (PAMs), must be present before CRISPR/Cas9 can cut. This PAM, also called the short sequence genome tag, sequence will not be present before the coding part of the DNA of the bacteria, but is present in the invading DNA from the pathogen (Sander J.D. et al., 2014).
Figure 8. A) Naturally occurring CRISPR/Cas9 system. CRISPR genome generates various crRNA with protospacers and tracrRNA. After association with Cas9 a DSB will lead to loss of the pathogenic nucleic acid, for example viral DNA. B) The most widely used CRISPR/Cas9 system uses a fusion protein between crRNA and tracrRNA, which will also form a complex with Cas9 and lead to DSB next to the PAM sequence. (Sander J.D. et al., 2014)
To genetically modify DNA with the CRISPR/Cas9 technique, two components must be present in the cell nucleus; Cas9 nuclease and single stranded guide RNA (sgRNA). The sgRNA is a fusion protein of the crRNA and the tracrRNA. 20 nucleotides at the 5’ of the sgRNA correspond to the protospacer, and are therefore responsible for directing the Cas9 to the target sequence, see Figure 8. It must immediately precede a PAM matching 5’-NGG in order to activate the nuclease activity of the Cas9, as explained above (Jinek M. et al., 2012). This system has therefore a limit of changing 20 amino acids before the NGG sequence, see Figure 9. With new techniques, large sgRNA libraries arise, encompassing multiple gRNAs for almost every gene in an organism.
12/30
Figure 9: The CRISPR-Cas9 nuclease containing with sgRNA. The sgRNA targets the PAM just in front of the sequence of interest. It unzips the DNA which is complementary to the sgRNA and the nucleases domain nick the DNA strand. The unzipped region can be maximally 20 amino acids long prior to the PAM (Genome Editing; the CRISPR/Cas9 system).
An important question is how reliable the CRISPR/Cas9 system is. Off-target cleavage can lead to NHEJ induced mutations. Mutations due to off-target mutations can be as high as 2-5% for the normal form of Cas9. To prevent off-target cleavage, Cas9 nickase is used. Nickases are only able to cut 1 strand of the DNA, resulting in a single strand break, see Figure 10 (Ott de Bruin et al., 2015). Two nickases targeting opposite strands are required to generate a DSB within the target DNA, also called a dual nickase CRISPR system. In order to obtain a pharmacological effect, two nicks must be made by Cas9n and two complementary repair RNA templates are needed to repair the mutation. The chance of two off-target nicks being generated close enough to form a DSB resulting in unwanted repair, is thus very low. The Cas9n is a100- fold more specificity relative to wild type Cas9 with one of the sgRNAs. (Sander J.D. et al., 2014) The efficacy of the Cas9n is the same as Cas9, Ann Ran F. et al showed that 80% of their embryotic cell where modified with CRISPR/Cas9n. The optimal concentration of Cas9n plasmids and sgRNA for efficient gene targeting is between 100 ng/uL to 3 ng/uL of Cas9n mRNA while maintaining the sgRNA levels at a 1:20 Cas9:sgRNA molar ratio (Ann Ran F. et al., 2013).
Figure 10: Schematic presentation of CRISPR/Cas9 and CRISPR/Cas9n. A) CRISPR/Cas9 is the most commonly used system. After PAM recognition and aligning of the sgRNA Cas9 introduces a DSB (presented by the flash), 3 bp upstream of the PAM sequence. B) Cas9n is a mutant form of Cas9 and doesn’t introduce DSBs but only single strand breaks. To introduce new DNA via the template two Cas9n are needed with both their own sqRNA. (Ott de Bruin et al., 2015)
13/30
When multiple mutations are present in the GBA gene, DSBs can additionally be exploited to mediate multiple deletions in the genome (Cong L. et al., 2013). In this way, the genome can be modified in 1 procedure, even if multiple mutations are present.
dit is slechts een klein stukje van t gehele report (ging over het ontwikkelen voor een nieuwe therapy voor Gaucher Disease m.b.v. Crispr/Cas9 nickase.
in principe is het een techniek, waarbij je kleine stukjes in het DNA kunt veranderen. meestal heb je bij bepaalde ziektes mutaties in het DNA, die slechts 1-2 baseparen verschillen van de gezonde variant. met crispr/cas kun je het dna '' breken'' , het slechte stukje er uit halen, en de goede versie terug doen. deze cellen kweek je dan later op ex vivo en voeg je daarna toe terug aan de patient, of je gebruikt een virale factor om deze verandering van dna aan al je cellen in je lichaam te geven. (een virale factor = virus mechanisme die niet zo schadelijk voor je is als een echt virus waar je ziek van wordt ;))