Multiplex Genome Editing

Last update: Sep 2020

Multiplex genome editing consists of targeting multiple DNA sequences at the same time. While CRISPR-Cas9 systems are usually employed to edit several genes only, the CRISPR-Cas12a method has the potential for performing more extensive multiplex genome editing. Another reason why Cas12a is used for this purpose rather than Cas9 is that in several studies, Cas12a was shown to be slightly more specific than Cas9 when identifying targeted sequences.

As discussed before, Cas12a, unlike Cas9, does not need a tracrRNA, as it possesses a ribonuclease site within its WED domain, which allows it to process pre-crRNA by itself. The fact that tracrRNA is not needed means that there is less information to store in the CRISPR locus, which results in a shorter nucleotide sequence coding this locus. Therefore, the gRNA of Cas12a is much shorter than that of Cas9. This turns out to be a useful feature. When we introduce a CRISPR construct into an organism, we usually use plasmids. A single plasmid can only store a limited sequence length before it becomes too unstable. Because the sequence coding Cas9 nuclease and the components of its gRNA are so long, we can usually only fit several gRNAs targeting several DNA sequences into the plasmid. However, given that the gRNA (crRNA) of Cas12a is way shorter than that of Cas9, we can fit many more crRNA sequences into the plasmid as a single crRNA construct. After Cas12a nuclease is formed, it can process the pre-crRNAs by itself (without needing RNase), and start targeting multiple sites.

The plasmid that allows the storage of many crRNA sequences, with a stabilizer tertiary RNA structure, was designed by a team at ETH Zurich in Switzerland and the University of Groningen in the Netherlands. It enables targeting of up to 25 sites, though it could be theoretically possible to increase the number of targets to a hundred or more in the future. The next section provides more details on this study.

Multiplexed genome editing has so far been demonstrated in mammalian cells and rice (by simplified single transcriptional unit CRISPR system using FnCas12a and LbCas12a). It makes it possible to investigate diseases with complex genetic profiles through editing a vast number of genes at once and studying their combined influence on phenotype.

One more useful characteristic of Cas12a is its temperature dependence. For example, one team employing CRISPR-Cas12a for genome editing of Drosophila melanogaster reported that for most crRNAs, LbCas12a (Lachnospiraceae bacterium) is inactive at 18°C, and only becomes active at 29°C. This characteristic makes temperature-controlled multiplex genome editing possible, at least in some species.