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Open Access Publications from the University of California

PAM Nucleotide Deviation with CRISPR-Cas9 and sgRNA

  • Author(s): Singh, Gurbinder
  • et al.

CRISPR-Cas9 is a system that it is not only revolutionary in expeditious sequencing of deoxyribonucleic acids (DNA) but also serves to induce genomic alterations within the sequence as well. The methodology that allows CRISPR to behave in such a manner is its ability to function in conjunction with ribonucleic acids (RNA). A facet of this RNA is its ability to scale from micro to macro organisms and by doing so, represents an observable deviation in its sequence. These sequential changes in RNA are observable in the form of protospacer adjacent motif (PAM) nucleotides which directly associate with the single guiding RNA (sgRNA) systems that direct DNA to the Cas9 enzyme. These PAM nucleotides are segments on RNA that are changed when DNA is altered and can be monitored. Cas9 is the protein component of CRISPR that is responsible for genome editing. However, the problem is that there hasn’t been conclusive evidence that demonstrates sgRNA will scale in complex genomes such as mice or humans. My objective is to observe how the PAM nucleotides behave in a multicellular Mammalia organism such as Mice to determine if RNA is directing DNA to the Cas9 enzyme and inducing genetic alterations. To observe these PAM nucleotides, the Mice genome will be subject to the introduction of Cas9 systems to be observed with PCR, gel electrophoresis, and Illumina sequencing. These are techniques that will yield data that signify if CRISPR is effective in altering mice genomes. With significant observable deviations in the PAM nucleotide sequences, the conclusion will be that CRISPR-Cas9 is able to induce alterations in complex genomes such as Mice and eventually humans.

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