1. Gene Editing

Gene editing is a type of genetic engineering. DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases. A nuclease is an enzyme capable of cleaving the bonds between the subunits of nucleic acids. The nucleases create specific double-strand breaks at target locations in the genome and harness the cell’s endogenous mechanisms to repair the break by natural processes of homologous recombination and nonhomologous end-joining. Technologies used to accomplish this are Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), Transcription Activator-Like Effector Nucleases (TALEN), and Zinc-Finger Nucleases (ZFN).

a) CRISPR

Screen Shot 2016-05-26 at 2.42.24 PMCRISPRs are segments of prokaryotic DNA, found in prokaryotes such as bacteria and archaea, which contain repeating base sequences separated by spacer DNA scavenged from previous viral infiltrations. CRISPR-associated genes (cas) are associated with CRISPR repeats and encode genes for nuclease proteins that can cut or unwind DNA. Together, the CRISPR/cas system provides an elementary adaptive immune system that detects and destroys foreign DNA from plasmids and phages. More recently, the CRISPR adaptive immune system has been shown to facilitate RNA-guided sequence-specific DNA cleavage using the cas subtype, cas9, which provides a new class of genome engineering and editing tools.

Cas9 plasmids are used to modify DNA within a cell. Inside the cell, the plasmid expresses cas9 enzymes, CRISPR RNA (crRNA), and trans-activated crRNA (tracrRNA). The tracrRNA binds to the cas9 protein and attaches to the crRNA to form the complete cas9 complex. The crRNA is the active region used to detect and cleave target DNA. Cas9 plasmids can be specially designed to express specific crRNA for precise gene editing.

Cas9 RNA is mRNA used to express cas9 enzymes in cells. Direct application of cas9 RNA in a cell removes the need for genomic integration of the cas9 protein in expression vectors.

Cas9 Lentivirus is used for gene editing in cells that are not easily transfected using plasmids. The cas9 Lentivirus is pre-packaged and ready-to-infect pseudoviral par15-013_img_smticles for expression of cas9 wild-type nucleases and mutant nickases for generation of cell lines stably expressing cas9. Instead of the lentivirus’ DNA, the envelope encloses the cas9 lentivector.

Multiplex gRNA are vectors that contain multiple gRNA sequences. The combination of crRNA and tracrRNA is denoted as gRNA. A crRNA sequence will only target a unique DNA sequence. However, multiple gRNA sequences can be combined in a vector to be used by cas9 enzymes to modify, or target, multiple individual segments of DNA.

Cas9 Detection is used to accurately monitor the expression of the cas9 enzyme. QPCR/RT-PCR primer sets can detect and quantitate the Cas9 mRNA transcript expression levels. The cas9 editing enzyme protein itself can be tracked using a cas9 polyclonal antibody. Amplicons in different primer sets can be used to detect cas9 nuclease, cas9 Nickase and cas9 double mutant at messenger level.