recent years, novel genome editing techniques have been developed. These techniques use bacterial nucleases which are guided by small RNA molecules to a specific site in the genome to cleave the DNA. This causes a double strand break. This break can be repaired in cells, but the most common system (non homologous end joining (NHEJ) is error prone and results in small insertions or deletions (InDels) of nucleotides at the site of the double strand break. The bacterial nucleases are now used to specifically edit the genome to our liking.
There are multiple methods to perform these genomic edits:
Nuclease type II used for genome editing. We work with Cas9 from S. pyogenes and several mutants of this nuclease.
Clustered regularly interspaced short palindromic repeats.
Cas9 are guided to a specific site in the genome by a combination of two RNA molecules. One which recognizes the target DNA and a second trans activation cr(CRISPR)RNA (TracrRNA). These have been combined into a single guide (sg) RNA for easy use of the system.
Many reports show that genome editing via Cas9 mediated mutation has off target effects where Cas9 does not only cut the desired sequence. The major off targets are indicated
Cas makes use of an RNA which recognizes the target DNA and a trans activation cr(CRISPR)RNA (TracrRNA). These have been combined into a small guide (sg) RNA.
First 7 nucleotides after PAM sequence, vital for correct binding of Cas9, total guide is required for correct nuclease activity.
Protospacer adjacent motif, Cas9 recognizes NGG, Cas9-VRQR-HF1 recognizes NGAN, Cpf1 recognizes NTTT.
To acquire knockouts/mutants of specific genes there are two Cas9 mediated mutation strategies:
To generate insertional mutants, cleavage with WT Cas9, and a subsequent homologous driven repair (HDR) mechanism to introduce a specific mutation or gene (or both) is used. Not only a guide RNA and Cas9 are introduced in the cells/embryos, but also a repair DNA molecule which has the specific mutation or insert. We have successfully used several repair constructs.
A strong benefit of the mouse model has always been the possibility to generate gene specific, endogenously regulated, reporter models. This is done by introducing a (fluorescent) reporter or Cre protein in the 3’UTR of a gene in combination with an IRES or a V2A sequence. This ensures that the transcript ratio between gene and reporter is equal and to an endogenous level. The 3’UTR of a gene is by definition AT rich. However, Cas9 makes use of a PAM sequence which is CG rich and therefore very unsuitable to target the 3’UTR of most genes. This can be circumvented by using alternative nucleases or Cas9 nuclease variants which recognize different PAM sequences. For instance, Cpf1 recognizes PAMs which are AT rich and makes a staggered cut.
CRISPR/Cas9 is not only used to make mutations or integrations in the genome, but can also be modified to regulate transcription. A kinase dead form of Cas9 is then used (Cas9d) which is unable to cut the DNA but is only recruited to the site recognized by the sgRNA. When Cas9 is then fused to activator proteins like VP16, transcription blocking proteins, like KREB or even (de)methylation enzymes transcription can be altered.