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. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

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. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (> 230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors, CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.

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 SMN1 loss. Approved therapies circumvent endogenous SMN regulation and require repeated dosing or may wane. We describe genome editing of SMN2, an insufficient copy of SMN1 harboring a C6>T mutation, to permanently restore SMN protein levels and rescue SMA phenotypes. We used nucleases or base editors to modify five SMN2 regulatory regions. Base editing converted SMN2 T6>C, restoring SMN protein levels to wild type. Adeno-associated virus serotype 9-mediated base editor delivery in Δ7SMA mice yielded 87% average T6>C conversion, improved motor function, and extended average life span, which was enhanced by one-time base editor and nusinersen coadministration (111 versus 17 days untreated). These findings demonstrate the potential of a one-time base editing treatment for SMA.

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 (R2 = 0.94) and fitness using short-read data from directed evolution experiments, with substantial improvements over related methods. We validate Evoracle on phage-assisted continuous evolution (PACE) and phage-assisted non-continuous evolution (PANCE) of adenine base editors and OrthoRep evolution of drug-resistant enzymes. Evoracle retains strong performance (R2 = 0.86) on data with complete linkage loss between neighboring nucleotides and large measurement noise, such as pooled Sanger sequencing data (~US$10 per timepoint), and broadens the accessibility of training machine learning models on gene variant fitnesses. Evoracle can also identify high-fitness variants, including low-frequency 'rising stars', well before they are identifiable from consensus mutations.

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

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar ß-globin (HBBG), a non-pathogenic variant4,5. 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 HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar ß-globin represented 79% of ß-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.

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. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.

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. ABE8e contains eight additional mutations that increase activity (kapp) 590-fold compared with that of ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disruption of regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by the introduction of an additional mutation in the TadA-8e domain. Finally, we show that ABE8e can efficiently install natural mutations that upregulate fetal hemoglobin expression in the BCL11A enhancer or in the the HBG promoter in human cells, targets that were poorly edited with ABE7.10. ABE8e augments the effectiveness and applicability of adenine base editing.

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. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.

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. This set of base editors improves the targeting scope of cytosine and adenine base editing.

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. It was then that the function of these DNA repeats, which were named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), was finally elucidated. Researchers found that CRISPR, when combined with its CRISPR-associated partner (Cas), is crucial for the functioning of the bacterial adaptive immune system against viral phage infection. CRISPR sequences can be transcribed into targeting RNA molecules, and Cas enzymes are guided by these RNAs to cut specific viral DNA loci, rendering resistance against the viral infection. Scientists realized that this natural bacterial immune response system could be engineered to become a powerful genome editing tool. Prior to CRISPR, existing genome editing tools such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) relied solely upon protein-DNA interactions to target an enzyme to specific DNA sequences. The design, engineering and evolution of proteins for various DNA sequences is difficult and time-consuming. In contrast, the CRISPR-Cas system uses Watson-Crick base pairing between a guide RNA and the target DNA to localize the complex to specific DNA sequences. This feature enables users to simply change an RNA sequence to match a DNA target to reposition the whole complex. Since then, numerous talented scientists have headed into this field. Within a single decade, they have developed the CRISPR-Cas system into a powerful genome editing tool and applied it to the editing of microorganisms, plants, animals and even human embryos. David R. Liu, Professor of Harvard University and the Broad Institute, and an investigator of the Howard Hughes Medical Institute, is one of them. One of his major contributions to the field is the development of 'base editing'. His group engineered the CRISPR system to transform it from being DNA scissors that cut DNA into specific DNA base pair rewriters that directly convert one base pair to a different base pair. This development opens the door to precision genome editing, raising the possibility of treating thousands of genetic diseases that are caused by single point mutations in the human genome. Here, David talks about this exciting time for genome editing.

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. We characterize determinants of base editing outcomes in human cells and establish that the formation of undesired products is dependent on uracil N-glycosylase (UNG) and is more likely to occur at target sites containing only a single C within the base editing activity window. We engineered CDA1-BE3 and AID-BE3, which use cytidine deaminase homologs that increase base editing efficiency for some sequences. On the basis of these observations, we engineered fourth-generation base editors (BE4 and SaBE4) that increase the efficiency of C:G to T:A base editing by approximately 50%, while halving the frequency of undesired by-products compared to BE3. Fusing BE3, BE4, SaBE3, or SaBE4 to Gam, a bacteriophage Mu protein that binds DSBs greatly reduces indel formation during base editing, in most cases to below 1.5%, and further improves product purity. BE4, SaBE4, BE4-Gam, and SaBE4-Gam represent the state of the art in C:G-to-T:A base editing, and we recommend their use in future efforts.

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 2.5-fold. Additionally, we engineered base editors containing mutated cytidine deaminase domains that narrow the width of the editing window from ∼5 nucleotides to as little as 1-2 nucleotides. We thereby enabled discrimination of neighboring C nucleotides, which would otherwise be edited with similar efficiency, and doubled the number of disease-associated target Cs able to be corrected preferentially over nearby non-target Cs.