Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

Abstract

CRISPR-Cas has revolutionized genome editing and has a great potential for applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region, and the presence of a PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

The colors of CRISPR-Cas Type V : Broadening the genome editing toolkit for Saccharomyces cerevisiae

“…Interestingly, in the cases where we observed targeted base editing, only the aimed nucleotide was modified. Most likely, this is due to a globally low activity of the MmCas12m base editor, which coincidentally minimizes the off-target activity of the fusion protein (473). At the same time, it can be related with the previously hypothesized unstable binding of MmCas12m to the DNA in yeast (Chapter 4).…”