Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors.

The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (i) Cas9 binding can potentially expose both DNA strands for 'capture' by the deaminase, a feature that is enhanced by guide RNA mismatches; (ii) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (iii) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer and (iv) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C > T and G > A transitions over >65 bp with significant potential for targeted gene diversification.

Controllable genome editing with split-engineered base editors.

DNA deaminase enzymes play key roles in immunity and have recently been harnessed for their biotechnological applications. In base editors (BEs), the combination of DNA deaminase mutator activity with CRISPR-Cas localization confers the powerful ability to directly convert one target DNA base into another. While efforts have been made to improve targeting efficiency and precision, all BEs so far use a constitutively active DNA deaminase. The absence of regulatory control over promiscuous deaminase activity remains a major limitation to accessing the widespread potential of BEs. Here, we reveal sites that permit splitting of DNA cytosine deaminases into two inactive fragments, whose reapproximation reconstitutes activity. These findings allow for the development of split-engineered BEs (seBEs), which newly enable small-molecule control over targeted mutator activity. We show that the seBE strategy facilitates robust regulated editing with BE scaffolds containing diverse deaminases, offering a generalizable solution for temporally controlling precision genome editing.

High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening.

CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells.

High-Throughput Screening of Catalytic H2 Production.

Hydrogenases, ferredoxins, and ferredoxin-NADP+ reductases (FNR) are redox proteins that mediate electron metabolism in vivo, and are also potential components for biological H2 production technologies. A high-throughput H2 production assay device (H2 PAD) is presented that enables simultaneous evaluation of 96 individual H2 production reactions to identify components that improve performance. Using a CCD camera and image analysis software, H2 PAD senses the chemo-optical response of Pd/WO3 thin films to the H2 produced. H2 PAD-enabled discovery of hydrogenase and FNR mutants that enhance biological H2 production is reported. From a library of 10 080 randomly mutated Clostridium pasteurianum [FeFe] hydrogenases, we found a mutant with nearly 3-fold higher H2 production specific activity. From a library of 400 semi-randomly mutated Oryza sativa FNR, the top hit enabled a 60 % increase in NADPH-driven H2 production rates. H2 PAD can also facilitate elucidation of fundamental biochemical mechanisms within these systems.

Human Germline CRISPR-Cas Modification: Toward a Regulatory Framework.

CRISPR germline editing therapies (CGETs) hold unprecedented potential to eradicate hereditary disorders. However, the prospect of altering the human germline has sparked a debate over the safety, efficacy, and morality of CGETs, triggering a funding moratorium by the NIH. There is an urgent need for practical paths for the evaluation of these capabilities. We propose a model regulatory framework for CGET research, clinical development, and distribution. Our model takes advantage of existing legal and regulatory institutions but adds elevated scrutiny at each stage of CGET development to accommodate the unique technical and ethical challenges posed by germline editing.

Chemical lysis of cyanobacteria.

We have developed a mixture of enzymes and chemicals that completely lyse cyanobacteria. Since the treatment involves only readily-available chemicals and simple proteins that degrade the components of the cyanobacterial cell wall, it can easily be used in high-throughput applications requiring lysis for subsequent intracellular measurements. Our lysis technique consistently enables complete lysis of several different cyanobacterial strains, and we demonstrated that DNA, mRNA, and proteins are preserved in the lysates. Chemical lysis can be superior to existing techniques because of its convenience, reliability, and amenability to a variety of downstream applications.