Gracie Duthie Biology 212 October 20th, 2022 The Relationship Between Phage Propagation and CRISPR Casesstes The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas immune system is present in many bacteria and most archaea. It is based on a natural method used by bacteria in protection against viruses. The CRISPR Cas system recognizes and cleaves foreign DNA or RNA. CRISPR Cas9 consists of two key molecules which cause a change in the DNA sequence, also known as a mutation. These two molecules include the guide RNA and the Cas9 enzyme. Guide RNA is a sequence that is able to find a specific sequence in the DNA and bind to it. It has bases that are complementary to the target DNA sequence bases therefore it will only bind to the target sequence region of the genome. Cas9 is a bacterial protein that defends bacteria against bacteriophage infections once the guide RNA recognizes the DNA sequence, the Cas9 enzyme cuts the DNA at the target location. When comparing the CRISPR method to the CRISPR Cas9 system, it can be identified that both processes need an enzyme that recognizes foreign sequences. After the DNA has been cut by Cas9, pieces of the genetic material are either added, or deleted, or other changes are made by replacing an existing DNA segment with a new one. Through this process, researchers are able to study specific genes. CRISPR can be found in the bacteria S. thermophilus which produces lactic acid and is used in the dairy industry
(Fernández, et al., 2017). Bacteriophages, also known as phages, are viruses that only infect bacteria. Many phages are able to infect the S. thermophilus bacteria. CRISPR may limit the growth of a bacteriophage. The relationship between the number of spacers on a bacterial cell and the sensitivity of the phage is unknown (Rodolph, et al., 2007). It is also still being determined where the RNA used by bacterial cells for protection originates from. It has been suggested that the RNA that cuts phage DNA molecules comes from past phage and plasmid infections (Bolotin, et al., 2005). It was also proposed that cut DNA is stored in the spacer of the CRISPR region of a bacterial cell. Although these suggestions have been made, research is still ongoing about what effect adding a greater amount of CRISPR cassettes and spacers will have on phage propagation and sensitivity. The results of this lab show demonstrate that when there is a more significant amount of spacers, there is more viral resistance. Through the use of several different types of S. thermophilus phage-resistant mutants, we can determine the number of spacers and identify how CRISPR spaces impact the resistance of the bacteriophages. This can be observed by infecting phage-resistant strains with a phage on agar plates and determining the resistance. Spacers can also be further studied by adding or removing them in a CRISPR sequence to see if they correspond with phage sensitivity (Barrangou, et al., 2007).
References 1. CRISPR provides acquired resistance against