CH391L/S14/CRISPR
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 randomnly or at precise five “letter” sequences. Surely this has been useful to edit DNA, but a sequence like TTGCC is frequent on stretches of DNA the length of genes, thus the nuclease that cleaves at this recognition sequence likely will cut all over a gene. This off-site nuclease activity limits the capability to use these enzymes (restriction enzymes) for directed gene modifications. 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. Riboswitches, for example, can be made to have high affinity to a specific genome site where it can be induced to perform gene modifications, but these and other DNA targeting molecules can be costly and challenging to make. 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 system that targets and cleaves DNA sequences. 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
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 of the foreign DNA (called spacers) get incorporated between short repeat sequences in CRISPR regions. The CRISPR loci are transcribed to produce CRISPR RNA (crRNA), which contains invader-specific complimentary sequences (called protospacer). But first, transcription of CRISPR produces a long CRISPR RNA or pre-crRNA containing the 22 nucleotide (nt) repeat sequences interspaced by the diverse 20-nt spacer sequences from the foreign DNA. Then a trans-activating crRNAs (tracrRNAs) binds crRNAs repeat sequences triggering the processing of CRISPR’s initial transcript by CRISPR associated proteins (Cas genes) and ribonuclease III (RNA pol III) into single spacer-repeat sequences mounted on a Cas protein endonuclease complex. Finally, the mature CRISPR/Cas complex is left with a single spacer sequence as guide-RNA to specifically target DNA. Once it detects the target foreign DNA, it binds, unwinds and cuts at the precise complimentary sequence if it also has the correct protospacer adjacent motif (PAM) at the 3’-end, resulting in the the silencing of the invader’s genes . Thus, in nature, the CRISPR/Cas system is essentially an adaptive bacterial defense system against viruses and foreign DNA. 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 has been preferentially used given that the only thing needed to target a DNA sequence is to modify the guide crRNA.
CRISPR/Cas gene editing
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.
