Gene Editing

Genome editing has emerged as one of the most exciting new areas of therapeutic development. A variety of tools exist for genome editing. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 has emerged as the most popular genome editing tool, due to its’ efficiency and ease of use. Co-transfection of Cas9 mRNA with a guide RNA results in a double stranded break that can be repaired by non-homologous end joining (NHEJ), leading to indels that inactivate target genes. If a donor DNA template with homology to the cut site is also co-transfected, homology directed repair can lead to gene correction or insertion.

TriLink offers three types of catalog mRNAs for CRISPR genome editing. For CRISPR/Cas9 gene editing, these include Cas9 mRNA with unmodified bases, as well as Cas9 mRNA modified with 5-methoxyuridine to reduce innate immune responses. We also offer Cas9 nickase modified with 5-methoxyuridine. Cas9 nickase has a D10A amino acid mutation that prevents cleavage of one of the DNA strands. As a result, the Cas9 Nickase generates a single stranded nick instead of a double stranded break. DNA cleavage or editing is directed to a specific chromosomal location by a single guide strand RNA (sgRNA) of ~100 nucleotides. An alternative nuclease for CRISPR is Cas12a. CRISPR/Cas12a gene editing uses a RNA guide strand of ~42 nucleotides. TriLink offers unmodified Cas12a or 5-methoxyuridine modified Cas12a mRNAs. 

Included in our gene editing tools are Cre recombinases, a tyrosine recombinase that catalyzes recombination between two loxP sites. Two separate DNA species that both contain loxP sites can undergo a fusion event as the result of Cre mediated recombination. Cre is useful for inserting DNA sequences into genomes that contain loxP sites. It is also useful for cell marking studies in which Cre expression activates an inactive reporter gene flanked by loxP sites in transgenic mouse lines. Cells transfected with Cre mRNA will then express the reporter and reveal which cells were productively transfected.

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