Paper of the month July 2019

Improved CRISPR-Cas12a-assisted one-pot DNA editing method enables seamless DNA editing

The CRISPR-Cas systems can be applied for fast but precise genome editing in essentially all cells. A short oligonucleotide guides the endonuclease to the correct genomic location and induces a break in the DNA. The CRISPR-Cas system can also be employed for molecular cloning of large plasmids, as reported in this article.

Molecular cloning consists mainly of two methods: restriction-ligation assembly (traditional cloning & Golden Gate) and homology-directed assembly (Gibson). Both methods face limitations and can be time consuming. In this article, the authors utilized the class II type V endonuclease Cas12a to perform large scale and precise cloning in a one-pot digestion and ligation reaction. The Cas12a enzyme leaves a 5 bp user defined sticky end after digestion and does not require external aid to process the nascent crRNA array into mature crRNA, nor does it utilize a tracrRNA. These characteristics make the enzyme suitable for molecular cloning. However, the need for an YTN (Y=C/T) PAM recognition site limits its use.

To increase the PAM recognition sites and by extension, increase the target range, the expression plasmid pET28-FnCas12a-TEV expressing wild type Francisella novicida Cas12a (FnCas12a) underwent mutagenesis. Residues around the PAM DNA duplex dictate selectivity and alteration to these recognition site residues produced 17 additional Cas12a variants. Each mutant had varying efficiency, some with an overall decreased cleavage capacity and others with an improved capacity. Two mutants stood out: EP16 and EP15. EP16 recognized 60 out of 64 possible PAMs and cleaved efficiently at YN targets, while EP15 recognized fewer PAM recognition sites but cleaved more efficiently in TAA, TGC and CTT. Together, they can efficiently target almost any sequence.

Therefore, Cas12a proves to be a viable restriction enzyme for usage in the improved CRISPR-Cas12a-assisted one-pot DNA editing (iCOPE). To assemble the digested and ligated product, the digestion enzyme needs to be specific and the recognition site abolished after ligation. In the article, the authors double-digested both vector and insert with FnCas12a, which destroyed the PAM recognition site. For specificity the crRNA is vital and traditionally crRNA in vitro transcription would take an additional day but in the iCOPE method the authors simply included RNA transcription simultaneously as digestion and ligation. In short, added reagents to the iCOPE method were: circle vector, linearized insert, in vitro transcription mix, Cas12a, dsDNA for crRNA transcription, RNase inhibitor and T4 DNA ligase.

Using the iCOPE method, almost 100% of the recombinants were correct and the assembly took 4-6h instead of one to two days, as is the case in classic molecular cloning.

In summary, with the new iCOPE method, the authors significantly increased possible target yield by mutating the FnCas12a enzyme and provided an alternative to the restriction-ligation or homology-directed assembly, giving it a broad potential.