Genomic engineering is the process of modifying specific sites in DNA sequences of cells or organisms. The ability to achieve effective and precise editing of the genome has been achieved and advanced through the development of sequence-specific nucleases1. Zinc-finger nucleases and transcription activator-like effector nucleases (TALENs) are programmable DNA binding nucleases previously used to achieve this targeted editing of DNA. However these tools have been found to be both time consuming and difficult to employ.
The development a system known as CRISPR/Cas9 system brings both ease and efficiency to world of targeted genome editing. The CRISPR/Cas9 system was discovered through the study of the adaptive immune response in bacteria. Clustered …show more content…
These crRNA act as a honing device, used to direct certain Cas enzymes to degrade target nucleic acids1. A second form of noncoding RNA, trans-activating RNA (tracrRNA), forms a complex with crRNA and the Cas endonuclease to locate and cleave the invading DNA. Cas 9 is a specific Cas gene with the ability to induce double stranded cleavage in the genomic DNA1. This Cas 9 endonuclease is one of the major components in the CRISPR/Cas9 system for targeted genome modification. The second major component is a crRNA-tracrRNA complex utilized to guide Cas9 to a specific site on the genome and induce double stranded cleavage (figure 1). This synthetic guide RNA complex is termed single guided RNA (sgRNA). Together, the Cas9 endonuclease and sgRNA form a complex with genomic DNA to specifically target sites on the genome. This genomic site is an approximately 20-base nucleotide sequence complementary to the sgRNA sequence adjacent to a protospacer adjacent motif (PAM). The creation of site-specific double stranded breaks by the system triggers genome editing through either nonhomologous end joining (NHEJ) or homology directed repair (HDR). Nonhomologous end
The development a system known as CRISPR/Cas9 system brings both ease and efficiency to world of targeted genome editing. The CRISPR/Cas9 system was discovered through the study of the adaptive immune response in bacteria. Clustered …show more content…
These crRNA act as a honing device, used to direct certain Cas enzymes to degrade target nucleic acids1. A second form of noncoding RNA, trans-activating RNA (tracrRNA), forms a complex with crRNA and the Cas endonuclease to locate and cleave the invading DNA. Cas 9 is a specific Cas gene with the ability to induce double stranded cleavage in the genomic DNA1. This Cas 9 endonuclease is one of the major components in the CRISPR/Cas9 system for targeted genome modification. The second major component is a crRNA-tracrRNA complex utilized to guide Cas9 to a specific site on the genome and induce double stranded cleavage (figure 1). This synthetic guide RNA complex is termed single guided RNA (sgRNA). Together, the Cas9 endonuclease and sgRNA form a complex with genomic DNA to specifically target sites on the genome. This genomic site is an approximately 20-base nucleotide sequence complementary to the sgRNA sequence adjacent to a protospacer adjacent motif (PAM). The creation of site-specific double stranded breaks by the system triggers genome editing through either nonhomologous end joining (NHEJ) or homology directed repair (HDR). Nonhomologous end