Difference between revisions of "CH391L/S14/CRISPR"

Revision as of 20:41, 29 March 2014

Background

Decades ago, the discovery of nucleases, enzymes that cut DNA, opened the door to DNA editing. While exonucleases cleave terminal nucleotides of a DNA strand, endonucleases cleave within a DNA strand and these can do so randomly, structure-specific or at precise short DNA sequences. Breakthroughs in our ability to edit genes have come in the form of techniques with better specificity, which means the targeting of a genomic sequence while excluding cleavage at other sites. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and homing meganucleases can be programmed to cleave at a specific genomic site, but these can be costly and challenging to make.

Summary

In 2007, it was discovered that certain bacteria had a defense system that combined short RNA guide oligonucleotides – virus or plasmid derived - with endonucleases. It was called the CRISPR/Cas system and it is an RNA-guided endonuclease that allows the cell to recognize and cleave DNA that it has previously encountered. Therefore, it acts as an adaptive immune system that protects a bacteria against invading DNA. Not much after its discovery, CRISPR/Cas systems in combination with recombination techniques were successfully used to modify genes in prokaryotes and plants. Very recently CRISPR/Cas gene editing has been shown to work even in human cells. The unprecedented versatility of the CRISPR/Cas system has empowered scientist with an easily programmable and surgically precise DNA editing tool.

How the CRISPR/Cas system works

The three stages of the CRISPR/Cas system (1) adaptation, (2) crRNA biogenesis and (3) invader silencing (interference).

• Adaptation

CRISPR stands for Clustered Regularly Inter-Spaced Palindromic Repeats, and these are genomic regions where prokaryotes are known to retain the footprint -sequence fragments- of previous encounters with virus or plasmid DNA. The way it works in nature is that any newly encountered invader that the bacteria survives gets cleaved and fragments from the foreign DNA (called spacers) get incorporated between short repeat sequences in CRISPR regions.

• crRNA Biogenesis

The CRISPR loci are transcribed to produce CRISPR RNA (crRNA), containing diverse repeat-spacer sequences which then are processed to separately become part of a CRISPR/Cas complex. During crRNA biogenesis, trans-activating crRNAs (tracrRNAs) bind to the repeat sequences in the newly transcribed long CRISPR’s transcript, triggering the transcript to be processed (by RNAse III) into the discrete space-repeat Cas bound sequences that make the mature CRISPR/Cas complexes. The spacer that remains in the CRISPR/Cas complex becomes the RNA-guide component set to detect the complementary sequence in invading DNA by guiding Cas nuclease domains to cleave at the specific target site (called protospacer).

• Invader silencing (gene interference)

Once a CRISPR/Cas system detects the target foreign DNA, it binds, unwinds and cuts at the precise complimentary sequence. It only requires the presence of a the protospacer adjacent motif (PAM). Thus, in nature, the CRISPR/Cas system is essentially an adaptive bacterial defense system against viruses and foreign DNA.

CRISPR/Cas gene editing

There are three types of CRISPR/Cas systems, but the main difference is that type I and type III systems depend on a large multi-Cas protein complex, while on type II systems only Cas9 is responsible for crRNA-guided silencing. It is for this reason that CRISPR/Cas9 type II systems have been preferentially used for DNA editing applications given that the only thing needed to target a DNA sequence is to modify the guide crRNA.

CRISPR-Cas system genome editing generally involves three steps: (1) site specific double-stranded DNA break (2) activated DNA repair machinery (3) Non-homologous end joining (NHEJ) or homologous recombination (HR). NHEJ results in nucleotide insertions and deletions at the DSB site. This allows the experimenter to add designed oligonucleotides to perform gene editing at the specific site.

Exciting CRISPR/Cas applications:

• Removal of a bacterial strain by use of genome targeting (Goma 2013).
• Proteins can be targeted to any dsDNA sequence by simply fusing them to Cas9 (Mali 2013)
• Targeted genome regulation

Advantages and Disadvantages of CRISPR/Cas editing

Advantages

• CRISPR/Cas systems are invader-specific, adaptive, and heritable.
• Easy to program targeted gene specific modifications using the CRISPR-Cas9 system, given it merely requires changing the sequence (20bp) of the guide RNA.
• Amenable to high-throughput construction of a library of targeting vectors - multiplexing.
• Works on all cell types.

Disadvantages

• Remaining offsite nuclease activity (off-target cleavage): up to six mismatches are tolerated between crRNA and target DNA.
• Efficiency: it can be improved.

iGEM

http://2013.igem.org/Team:Freiburg/Team2

References