Genome Editing: Overview Of CRISPR System

Microorganism have invented various strategies to allow them to survive exposure to foreign DNA. This is an adaptive system prokaryote’s have adopted and based on a region of DNA called Clustered Regularly Interspaced Short Palindromic Repeats (or CRISPR). CRISPR is a system used by Bacteria and Archaea to recognise foreign DNA and distinguish between foreign and cell DNA. The CRISPR loci consists of several direct repeats that are separated by variable sequences called spacers. These spacers are often adjacent to Cas genes which encode for a large family of proteins. CRISPRR combines with the cas9 gene to form a system.

Scientists have identified that the spacer regions correlate to phage genomic DNA. Thus, when new foreign DNA is encountered new spacers will be added to CRISPR, this makes CRISPR a chronological record of all the viruses the cell has come across. There are three defined stages of the CRISPR-cas9 system. The first stage is adaptation, this stage leads to a new spacer that will be inserted into the CRISPR locus. The second stage is expression, this stage includes the action of the Cas genes (these can be helicases and nucleases). The cas9 gene will transcribes CRISPR into a long precursor CRISPR RNA which is termed pre-creRNA (pre-creRNA can then be changed into creRNA). The final stage is interference, in this stage the crRNA recognises and targets nucleic acids which are then removed from the cell. This done by the crRNA which guides the cas9 protein to the foreign DNA which is then cleaved by cas9. The cleavage site is 3-4 sequences upstream of the PAM sequence and cleavage results in a double-strand break.

The three main components of CRISPR are the cas9 gene, crRNA and the tracrRna. The crRNA guides the Cas9 enzyme to a specific DNA sequence. The Cas9 enzyme then cleaves the target sequence. The PAM sequence mediates the binding of the Cas9 endonuclease to the target genomic locus. The PAM sequence enables the Cas9 enzyme to perform a double stranded cut. The PAM sequence is a 3-base pair sequence and is found immediately downstream of the target locus. crRNA’s are transcribed form the CRISPR locus, the crRNA guide the Cas9 complex to the target DNA. crRNA is usually between 1-31 nucleotides long and binds to the Cas9 enzyme (crRNA occurs naturally in the cell wile gRNA is artificial crRNA). tracrRNA stand for trans-activating crRNA. One pathway for CRISPR activation uses tracrRNA’s, these RNA’s play a role in maturing crRNA’s. An RNA duplex is formed because tracrRNA is partially complementary to pre-crRNA, this induces base pairing between the two.

The RNA duplex is cleaved by RNaseIII and forms a crRNA/tracrRNA hybrid (which contains only one spacer). The hybrid then bind to Cas9 and guides Cas9 to the target sequence. Reference for question 2Karvelis T. , Gasiunas G. , Miksys A. , Barrangou R. , Horvath P. and Siksnys V. , 2013. crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. US National Library of Medicine National Institute of Health, 10(5) pp. 841-51. Question 3Once CRISPR has been used to mutate an organism, the edited genome must be able to be identified. One can use screens or selections to identify edited genomes. A genetic screen is used to detect a genetic mutant. If the edited genome produces a specific phenotype, the progeny can then be screened for the specific phenotype. Screening can also take place for molecular lesions; these lesions are generated by editing the genome. An example of molecular lesion detection is restriction fragment length polymorphisms (RELP). RELP notices the differences in restriction sites between the wild-type DNA and the edited genome.

The basics behind RELP is that the region of interest where the mutation should have taken place will be visualised using gel electrophoresis, if the PCR product is not cut then the mutation did take place. A selection method could also be used. This can make use of lethal mutations, this means that only individual with a specific mutation (induced by using CRISPR) will survive a specific condition. Another example of a selection method uses drug resistance. The edited cell will have a rug resistance gene incorporated into their genome, thus only the cells that survive after the drug has been administered will be the desired edited cells and no screening will have to take place. Reference for question 3Thurtle-Schmidt and Te-Wen Lo, 2018. Molecular biology at the cutting edge: A review on CRISPR/Cas9 gene editing for undergraduates. US National Library of Medicine National Institute of Health, 46(2) pp. 195-205.