Prime editing using CRISPR-Cas12a and circular RNAs in human cells.

Genome editing with prime editors based on CRISPR-Cas9 is limited by the large size of the system and the requirement for a G/C-rich protospacer-adjacent motif (PAM) sequence. Here, we use the smaller Cas12a protein to develop four circular RNA-mediated prime editor (CPE) systems: nickase-dependent CPE (niCPE), nuclease-dependent CPE (nuCPE), split nickase-dependent CPE (sniCPE) and split nuclease-dependent CPE (snuCPE). CPE systems preferentially recognize T-rich genomic regions and possess a potential multiplexing capacity in comparison to corresponding Cas9-based systems.

Assigning functionality to cysteines by base editing of cancer dependency genes.

Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation.

Strand-preferred base editing of organellar and nuclear genomes using CyDENT.

Transcription-activator-like effector (TALE)-based tools for base editing of nuclear and organellar DNA rely on double-stranded DNA deaminases, which edit substrate bases on both strands of DNA, reducing editing precision. Here, we present CyDENT base editing, a CRISPR-free, strand-selective, modular base editor. CyDENT comprises a pair of TALEs fused with a FokI nickase, a single-strand-specific cytidine deaminase and an exonuclease to generate a single-stranded DNA substrate for deamination.

Discovery of deaminase functions by structure-based protein clustering.

The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities.

Precise integration of large DNA sequences in plant genomes using PrimeRoot editors.

A technique for chromosomal insertion of large DNA segments is much needed in plant breeding and synthetic biology to facilitate the introduction of desired agronomic traits and signaling and metabolic pathways. Here we describe PrimeRoot, a genome editing approach to generate targeted precise large DNA insertions in plants. Third-generation PrimeRoot editors employ optimized prime editing guide RNA designs, an enhanced plant prime editor and superior recombinases to enable precise large DNA insertions of up to 11.

Base editing rescue of spinal muscular atrophy in cells and in mice.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, arises from survival motor neuron (SMN) protein insufficiency resulting from loss. Approved therapies circumvent endogenous SMN regulation and require repeated dosing or may wane. We describe genome editing of , an insufficient copy of harboring a C6>T mutation, to permanently restore SMN protein levels and rescue SMA phenotypes.

A molecular glue approach to control the half-life of CRISPR-based technologies.

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life.

Tuning plant phenotypes by precise, graded downregulation of gene expression.

The ability to control gene expression and generate quantitative phenotypic changes is essential for breeding new and desired traits into crops. Here we report an efficient, facile method for downregulating gene expression to predictable, desired levels by engineering upstream open reading frames (uORFs). We used base editing or prime editing to generate de novo uORFs or to extend existing uORFs by mutating their stop codons.

Reconstruction of evolving gene variants and fitness from short sequencing reads.

Directed evolution can generate proteins with tailor-made activities. However, full-length genotypes, their frequencies and fitnesses are difficult to measure for evolving gene-length biomolecules using most high-throughput DNA sequencing methods, as short read lengths can lose mutation linkages in haplotypes. Here we present Evoracle, a machine learning method that accurately reconstructs full-length genotypes (R = 0.

Base editing of haematopoietic stem cells rescues sickle cell disease in mice.

Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB. We used a custom adenine base editor (ABE8e-NRCH) to convert the SCD allele (HBB) into Makassar β-globin (HBB), a non-pathogenic variant. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBB to HBB.

DNA capture by a CRISPR-Cas9-guided adenine base editor.

CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex.

Author Correction: Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.

Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), which resulted in ABE8e.

An anionic human protein mediates cationic liposome delivery of genome editing proteins into mammalian cells.

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts.

Author Correction: Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors.

Base editing requires that the target sequence satisfy the protospacer adjacent motif requirement of the Cas9 domain and that the target nucleotide be located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG protospacer adjacent motifs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4-5 nucleotides to up to ~8-9 nucleotides and reduced byproduct formation.

An exciting time for genome editing: an interview with David R. Liu.

In 1987, several Osaka University researchers discovered a special kind of clustered DNA repeats in bacteria. Within a few years, two other groups independently discovered the same phenomenon but no one knew its function at the time. Only a small handful of scientists studied this property from its discovery in 1987 to 2005.

Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity.

We recently developed base editing, the programmable conversion of target C:G base pairs to T:A without inducing double-stranded DNA breaks (DSBs) or requiring homology-directed repair using engineered fusions of Cas9 variants and cytidine deaminases. Over the past year, the third-generation base editor (BE3) and related technologies have been successfully used by many researchers in a wide range of organisms. The product distribution of base editing-the frequency with which the target C:G is converted to mixtures of undesired by-products, along with the desired T:A product-varies in a target site-dependent manner.

Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.

Base editing induces single-nucleotide changes in the DNA of living cells using a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair. This genome editing approach has the advantage that it does not require formation of double-stranded DNA breaks or provision of a donor DNA template. Here we report the development of five C to T (or G to A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing 2.

Tertiary Amine Pyrazolones and Their Salts as Inhibitors of Mutant Superoxide Dismutase 1-Dependent Protein Aggregation for the Treatment of Amyotrophic Lateral Sclerosis.

Pyrazolone derivatives have previously been found to be inhibitors of Cu/Zn superoxide dismutase 1 (SOD1)-dependent protein aggregation, which extended survival of an amyotrophic lateral sclerosis (ALS) mouse model. On the basis of ADME analysis, we describe herein a new series of tertiary amine-containing pyrazolones and their structure-activity relationships. Further conversion to the conjugate salts greatly improved their solubility.

Proteasome activation is a mechanism for pyrazolone small molecules displaying therapeutic potential in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action.