Introduction to CRISPR Cas9 technology. View in slide show after downloading for better viewing. Description is minimal, but it will be worth going through the slides that are full of pictures, if you have a minimal understanding of CRISPR.
Prepared in Oct 2015
7. TALENS: Transcription Activator-Like
Effector Nucleases
DNA binding domain: 33-34 amino acid sequence
•Secreted by Xanthomonas when they infect
various plant species
•Bind promoter sequences in the host plant
•Activate the expression of plant genes that
aid bacterial infection
8. Meganucleases or Homing
Endonucleases:
5 families:
LAGLIDADG
GIY-YIG
HN
His-Cys box
PD-(D/E)XK
5 families:
LAGLIDADG
Recognizes 14-40 bp long sequences
9. Protein based gene editing systems are difficult to
engineer
Expensive
Time consuming
Off-targeting
Alternative: CRISPR/Cas9 System
25. CRISPR/Cas9
Advantages
Based on Watson-Crick base-pairing of sgRNA-DNA and an NGG PAM motif
straightforward and flexible
Multiplex gene targeting
Much larger targetable sequence space
Limitations
The requirement of a protospacer adjacent motif (PAM) sequence limits the
number of potential target sequences
Sequence specificity to target loci is only 14 nt long (12 nt of sgRNA and 2nt of the
PAM), which can recur around ~11 times in a human genome
Endogenous chromatin states and modifications may prevent the sequence specific
binding
27. Hereditary tyrosinemia Type I
Fumaryl-acetoacetate hydrolase (FAH)
Homozygous GA mutation in the last exon
Liver failure and death in infancy
FOK1 cuts DNA once it is bound to the DNA. Generally, this binding is favoured by the palindromic sequence in the restriction site. In ZFN and TALENS, the binding is favoured by the DNA binding domains of ZFN and TALENS
repeat-variable diresidue (RVD)
Whereas Type II restriction enzymes bind short, usually symmetric, recognition sequences of 4 to 8 bp, homing endonucleases bind very long and in many cases asymmetric recognition sequences spanning 12 to 40 bp
2A Peptides:
These peptides, first discovered in picornaviruses, are short (about 20 amino acids) and produce equimolar levels of mulitple genes from the same mRNA. The term "self-cleaving" is not entirely accurate, as these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream.4 The "cleavage" occurs between the Glycine and Proline residues found on the C-terminus meaning the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the Proline.
The length of the repeat can vary from 21bp to 48 bp, whereas spacers are typically between 26 bp and 72 bp
HNH is a domain in the active site
RuvC domain can be inactivated by a D10A mutation and the HNH domain can be inactivated by an H840A mutation.
The KRAB domain, which is found in the amino-terminal region of the proteins, behaves as a transcriptional repressor domain by binding to corepressor proteins, whereas the C2H2 zinc-finger motifs bind DNA.