While rDNA technologies are used with relative ease to produce genetically modified unicellular organisms, the process becomes increasingly difficult to accomplish in higher organisms. This is where CRISPR/ Cas9 genome editing comes in. As mentioned in Chapter 1, naturally occurring CRISPR elements confer microbial25 resistance to viral invasion (Barrangou et al. 2007). Upon invasion, small fragments of viral DNA are captured and integrated into the bacterial genome (Barrangou et al. 2007). Later invasion of a genetically similar virus results in transcription of the viral-associated CRISPR element. The mRNA produced guides a CRISPR-associated nuclease to hone in on and destroy complementary invading viral DNA by double-strand breaks. One of the simplest CRISPR-associated nucleases is Cas9.26 Using synthetic guide RNA (gRNA), recombinant CRISPR/Cas9 systems can be used to perform site-specific manipulation of DNA in all kinds of cells. gRNA directs Cas9 to a target site, where it produces double-strand breaks (see Figure 2.8). These breaks are resolved via endogenous nonhomologous end-joining or homologous-directed DNA repair (Zhang, Wen, and Guo 2014).

Coexpression or comicroinjection of Cas9/gRNAs into cells results in the deletion of target loci (Zhang, Wen, and Guo 2014). Deletions are clean—unmarked27-and if desired, in frame. If a ss DNA fragment with homology to the Cas9 cleavage site is introduced alongside the CRISPR machinery, the cell will use it as a template to repair the lesion (Zhang,

Wen, and Guo 2014). Unmarked allelic exchange is the result. What does this mean? For the first time, site-directed and precise genome editing (i.e., deletions, insertions, replacements) can be done quickly and easily in the context of whole cells or organisms. When performed on embryonic cells (or poultry germ cells), the organisms that develop are precisely genetically modified (Tizard et al. 2016). As such, there are major implications for studying and curing complex diseases as well as for the development of GMOs.

CRISPR/Cas9-mediated genome editing

Figure 2.8. CRISPR/Cas9-mediated genome editing.

< Prev   CONTENTS   Source   Next >