Characterization of the AcrIIC1 anti‒CRISPR protein for Cas9‒based genome engineering in E. coli.

Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli. We then demonstrate that the AcrIIC1 protein from Neisseria meningitidis robustly inhibits their DNA cleavage activity, but not their DNA binding capacity. Finally, we exploit these AcrIIC1:Cas9 complexes for gene silencing and base-editing, developing Acr base-editing tools. With these tools we pave the way for future engineering applications in mesophilic and thermophilic bacteria combining the activities of Acr and CRISPR-Cas proteins.

Efficient Genome and Base Editing in Human Cells Using ThermoCas9.

Most genetic engineering applications reported thus far rely on the type II-A CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpyCas9), limiting the genome-targeting scope. In this study, we demonstrate that a small, naturally accurate, and thermostable type II-C Cas9 ortholog from Geobacillus thermodenitrificans (ThermoCas9) with alternative target site preference is active in human cells, and it can be used as an efficient genome editing tool, especially for gene disruption. In addition, we develop a ThermoCas9-mediated base editor, called ThermoBE4, for programmable nicking and subsequent C-to-T conversions in human genomes. ThermoBE4 exhibits a three times larger window of activity compared with the corresponding SpyCas9 base editor (BE4), which may be an advantage for gene mutagenesis applications. Hence, ThermoCas9 provides an alternative platform that expands the targeting scope of both genome and base editing in human cells.

(R)evolution-on-a-chip.

Billions of years of Darwinian evolution has led to the emergence of highly sophisticated and diverse life forms on Earth. Inspired by natural evolution, similar principles have been adopted in laboratory evolution for the fast optimization of genes and proteins for specific applications. In this review, we highlight state-of-the-art laboratory evolution strategies for protein engineering, with a special emphasis on in vitro strategies. We further describe how recent progress in microfluidic technology has allowed the generation and manipulation of artificial compartments for high-throughput laboratory evolution experiments. Expectations for the future are high: we foresee a revolution on-a-chip.