Hyperactive Nickase Activity Improves Adenine Base Editing.

Base editing technologies enable programmable single-nucleotide changes in target DNA without double-stranded DNA breaks. Adenine base editors (ABEs) allow precise conversion of adenine (A) to guanine (G). However, limited availability of optimized deaminases as well as their variable efficiencies across different target sequences can limit the ability of ABEs to achieve effective adenine editing. Here, we explored the use of a TurboCas9 nickase in an ABE to improve its genome editing activity. The resulting TurboABE exhibits amplified editing efficiency on a variety of adenine target sites without increasing off-target editing in DNA and RNA. An interesting feature of TurboABE is its ability to significantly improve the editing frequency at bases with normally inefficient editing rates in the editing window of each target DNA. Development of improved ABEs provides new possibilities for precise genetic modification of genes in living cells.

Mutational rescue of the activity of high-fidelity Cas9 enzymes.

Programmable DNA endonucleases derived from bacterial genetic defense systems, exemplified by CRISPR-Cas9, have made it significantly easier to perform genomic modifications in living cells. However, unprogrammed, off-target modifications can have serious consequences, as they often disrupt the function or regulation of non-targeted genes and compromise the safety of therapeutic gene editing applications. High-fidelity mutants of Cas9 have been established to enable more accurate gene editing, but these are typically less efficient. Here, we merge the strengths of high-fidelity Cas9 and hyperactive Cas9 variants to provide an enzyme, which we dub HyperDriveCas9, that yields the desirable properties of both parents. HyperDriveCas9 functions efficiently in mammalian cells and introduces insertion and deletion mutations into targeted genomic regions while maintaining a favorable off-target profile. HyperDriveCas9 is a precise and efficient tool for gene editing applications in science and medicine.

Computationally designed hyperactive Cas9 enzymes.

The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.