Paper of the month May 2018

Tild-CRISPR Allows for Efficient and Precise Gene Knockin in Mouse and Human Cells.

CRISPR it’s a powerful tool for precise gene editing and due to its ease of use and low cost in terms of time and resource, it stood out as the main genetic editing tool. The precise modification of the DNA sequence– defined hereafter as knock in – like the correction of mutation involved in severe human diseases, it’s one of the most exiting aspect of this technology. However, the low knock in efficiency, has been one of the major weakness of this system. In this paper, Zi-jiang Chen and colleagues suggest a simple, but robust, strategy for enhanced Knock-in efficiency in vitro and in vitro. To perform genetic knock-in, the DNA has to undergo a double-strand break in the position in which the correction it is wanted to be inserted. CRISPR it is ideal for this task, thanks to its precision in cutting the DNA at the desired position. Also, together with CRISPR, a DNA strand is supplied. This DNA strand is constituted by:

• a central region: which harbours the modification to be inserted
• two flanking regions: those two are designed to have the same sequence of the regions surrounding the the double strand break area and thereby are called homology arms.

The DNA strand showing this layout can be used by the cell as template to repair the DNA break. So, by taking advantage of the inner DNA repair mechanism of the cell, the researcher can introduce the desired modification into the DNA.

So far, the DNA repair template was supplied as:

• a plasmid as
• a double strand DNA whose homology arms are surrounded by nucleotide stretches defined as junk sequences as
• a single strand DNA

The authors of the paper employ a double strand DNA previously digested in vitro - with restriction enzymes – to create a donor DNA with 800 bp homology arms on each side and lack the junk sequences. They call this layout Tild (Targeted Intergradation with Linearized dsDNA ) To test their approach as first experiment in vitro they decide to modify the Actb gene into mouse blastocyst. They supply a donor DNA so that the the sequence expressing the red fluorescent protein mCherry can be inserted within the sequence of the Actb. This way, if a red fluorescence is observed, in the blastocysts, it means that the knock-in was successful. Indeed, they could achieve a knock-out efficiency of 33,2% while the other approach showed an efficiency between 0% and 22%. As experiment in vivo they showed they could knock-in the mCherry protein by delivering their construct to the embryonic mouse brain and the latter was examined for mCherry signals seven day after. They showed that around 17% of the cells showed the mCherry signals, while with the other approaches (plasmid, single strand DNA  they could achieve only around 0,8% and 9%.

As final experiment, they tried to see of this enhancement in the knock-in efficiency could be also observed in human. To this end, they performed knock in  human embryos. In particular they injected Cas9 as mRNA, the gRNA and the DNA repair sequence in triplonuclear zygotes. In this case they targeted the integration of the green fluorescent protein (GFP) within the sequence of the gene Oct4. 10 out of 14 edited embryo, showed a successful knock in, confirming the applicability of this technique even in human.

The paper reports a straightforward and simple approach for improved knock-in in vitro but also in vivo. The method reported seems to overcome the low editing efficiency presented by the previous approaches, showing to be simple and flexible, since it was showed to be applicable to different targets. In conclusion we suggest reading in more details this paper, since the evidences presented may lead the way to further improvements in CRISPR techniques as well as helping the development of therapies.