The method provides 90 percent efficacy in gene editing, due to improved delivery, compared to other methods such as AAV.

“The lipid nanoparticles encapsulate messenger RNA (mRNA) encoding Cas9. Once the contents of the nanoparticles – including the sgRNA – are released into the cell, the cell’s protein-making machinery takes over and creates Cas9 from the mRNA template.” (Rees 2019)¹⁾

• Could be made with low-toxicity
• No limit for cargo size
• Immune response much less likely given non-viral nature

“CRISPR processes are still in their infancy, as the current tools are effective at cutting DNA but can result in random repair.” (Rees 2019)²⁾ Also, despite episomes or alternatively, the use of guide-RNA, offsite non-homologous integration (Deyle and Russell 2009)³⁾ has been an issue due to its viral background.

CRISPR is the most popular because of its ease and speed of use. It only takes one day to prepare CRISPR for an experiment.

Cas 9 enzyme has a sequence locator called the PAM. When the PAM finds a piece of DNA with its sequence, the enzyme unzips the adjacent DNA to see if it matches the guide-RNA. Matching DNA triggers the Cas 9 enzyme to double-strand-break (DSB) the DNA. A DSB is often not a clean break. Some nucleotides can be lost in the process.

The host DNA repair mechanisms come about to repair the DSB, and if a homologous DNA strand is around, it will be used as a template for repair. The homologous DNA can be provided as part of the technological payload, for the purpose of altering the genome.

Cas9 can also be engineered without DNA-cleaving and purposed for epigenome editing instead.

“Another development for CRISPR technologies came from researchers at Duke University in the US. The team successfully used Class 1 CRISPR systems for the first time to edit the epigenome of human cells. Conventional CRISPR-Cas9 methods are categorised as Class 2 systems.

The Class 1 technique makes use of multiple proteins in a process called CRISPR-associated complex for antiviral defence (Cascade). This complex binds with high accuracy to the correct sites. After binding, Cascade utilises a Cas3 protein to target and edit the DNA. They were also able to both activate and repress target gene expression.” (Rees 2019)⁴⁾

“Relying on homology-directed-repair (HDR) for editing risks introducing indels or chromosomal translocations. Even with a precisely targeted nuclease, with HDR, “you’re at the mercy of the cell,” Stoddard observes. For editing without the unpredictability of HDR, he adds, watch for developments in site-specific recombinases (SSRs).” (Tachibana 2019)⁵⁾

However, it can take 100 days to prepare an SSR.