It’s appearing all over the place : CRISPR/Cas9. The method has boomed since its 2012 breakthrough moment. Adam Mol walks us through this technology.
Currently, more and more discussions are appearing about CRISPR/Cas9. This technology can be used to edit the genome of almost any organism and the most controversial seems to be editing the genes of human embryos. How does this system works? CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) sequences and CRISPR-associated (Cas) genes were found in a wide variety of bacteria and archaea. The enzyme Cas9 is a DNA endonucleas
e found in many bacteria, where it functions as part of a defense system against invading DNA molecules, e.g. from viruses. The CRISPR/Cas9 system allows for specific genome disruption and replacement in a flexible and simple system, resulting in high specificity and low cell toxicity (Harrison et al., 2014; Peng et al., 2015).
Fig. 1 CRISPR/Cas9 in action (from Pennisi, 2013; modified)
The CRISPR/Cas9 genome editing system requires the co-expression of a Cas9 protein with a guide RNA. The crRNA and tracrRNA can be fused together to create a chimeric, single-guide RNA (sgRNA). Cas9 has two active sites that each cleave one strand of a double-stranded DNA molecule. The enzyme is guided to the target DNA by an sgRNA that contains a sequence that matches the sequence to be cleaved, which is demarcated by PAM (protospacer-adjacent motif) sequence. (There also exists a RNA-targeting CRISPR/Cas9 complex, called RCas9, that can edit RNA! by J. Doudna; O’Connell et al., 2014).
Fig. 2 Schematic of the RNA-guided Cas9 nuclease (from Ran et al., 2013)
In the CRISPR-Cas9 system derived from Streptococcus pyogenes the 20-bp target DNA must be followed at their 3’ends by 5’-NGG PAM, which can occur in the top or the bottom strand of genomic DNA. RNA-guided Cas9 activity creates site-specific double-stranded DNA breaks. Alternative modifications of Cas9 also exist, like Cas9 nickase (Cas9n). In this approach, pairs of Cas9n are targeted to generate single-strand breaks on opposite strands of the genomic target DNA. When the double-stranded breaks are repaired by standard cellular repair mechanisms, either by Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ) pathways the sequence at the repair site can be modified or new genetic information inserted.
The CRISPR/Cas9 system has been successfully used in bacteria, fungi, viruses, parasites, plants, animals, and human cell lines.
In the absence of a repair template, DSBs are re-ligated through the NHEJ process, which leaves scars in the form of insertion/deletion (indel) mutations. HDR typically occurs at lower and substantially more variable frequencies than NHEJ and the addition of donor DNA (repair template) enables new sequence information to be inserted at the break site (Ran et al., 2013; Harrison et al., 2014; Peng et al., 2016).
Fig. 3 Double-Stranded Breaks (DSBs) repaired by Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) mechanisms (from Ran et al., 2013; modified)
The CRISPR/Cas9 system has been successfully used in bacteria, fungi, viruses, parasites, plants, animals, and human cell lines. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, scientists (including me!) have realized that this technology was not as easy as thought! There are several aspects that affect CRISPR/Cas9 efficiency and specificity, including Cas9 activity, target site selection and sgRNAs design, delivery system, off-target effects, and the incidence of HDR. Additional improvements of the CRISPR/Cas9 still are necessary (Harrison et al., 2014; Liang et al., 2015; Peng et al., 2016).
Fig. 4 Components required for CRISPR/Cas9 technology (from Harrison et al., 2015)
Finally, I would like to add that the common white button mushroom has been modified to resist browning and the US Department of Agriculture will not regulate a mushroom genetically modified with the CRISPR/Cas9 technology – making it the first CRISPR-edited organism to receive a green light from the US government (Waltz, 2016).
> Harrison et al., 2014. A CRISPR view of development. Genes & Development 28:1859-1872
> Liang et al., 2015. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. Journal of Biotechnology 208: 44-53
> O’Connell et al., 2014. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516: 263-266
> Peng et al., 2016. Potential Pitfalls of CRISPR/Cas9-mediated Genome Editing. FEBS J. 283: 1218-1231
> Pennisi, 2013. The CRISPR Craze. Science 341: 833-836
> Ran et al., 2013. Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8: 2281-308
> Waltz, 2016. Gene-edited CRISPR mushroom escapes US regulation. Nature 532: 293