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The development of CRISPR-derived genome editing technologies has enabled the precise manipulation of DNA sequences within the human genome. In this review, we discuss the initial development and cellular mechanism of action of CRISPR nucleases and DNA base editors. We then describe factors that must be taken into consideration when developing these tools into therapeutic agents, including the potential for unintended and off-target edits when using these genome editing tools, and methods to characterize these types of edits. We finish by considering specific challenges associated with bringing a CRISPR-based therapy to the clinic, including manufacturing, regulatory oversight, and considerations for clinical trials that involve genome editing agents.

Base editors are fusions of a deaminase and CRISPR-Cas ribonucleoprotein that allow programmable installment of transition mutations without double-strand DNA break intermediates. The breadth of potential base editing targets is frequently limited by the requirement of a suitably positioned Cas9 protospacer adjacent motif. To address this, we used structures of Cas9 and TadA to design a set of inlaid base editors (IBEs), in which deaminase domains are internal to Cas9. Several of these IBEs exhibit shifted editing windows and greater editing efficiency, enabling editing of targets outside the canonical editing window with reduced DNA and RNA off-target editing frequency. Finally, we show that IBEs enable conversion of the pathogenic sickle cell hemoglobin allele to the naturally occurring HbG-Makassar variant in patient-derived hematopoietic stem cells.

The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5-A7 and ~3.2× higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2 with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification, which is maintained when multiplexed across three loci. Delivered as messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

The targeting scope of Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants is largely restricted to protospacer-adjacent motif (PAM) sequences containing Gs. Here, we report the evolution of three new SpCas9 variants that collectively recognize NRNH PAMs (where R = A or G and H = A, C, or T) using phage-assisted non-continuous evolution (PANCE), three new phage-assisted continuous evolution (PACE) strategies for DNA binding, and a secondary selection for DNA cleavage. The targeting capabilities of these evolved variants and SpCas9-NG were characterized in HEK293T cells using a library of 11,776 genomically integrated protospacer-sgRNA pairs containing all possible NNNN PAMs. The evolved variants mediate indel formation and base editing in human cells and enable the A•T-to-G•C base editing of a sickle-cell anemia mutation using a previously inaccessible CACC PAM. These new evolved SpCas9s, together with previously reported variants, in principle enable targeting the majority of NR PAM sequences and substantially reduce the fraction of genomic sites that are inaccessible by Cas9-based methods., Editorial summary PAM sequences without Gs can be edited with SpCas9 variants that were continuously evolved in the laboratory.

Cytosine base editors (CBEs) enable efficient, programmable reversion of T•A to C•G point mutations in the human genome. Recently, cytosine base editors with rAPOBEC1 were reported to induce unguided cytosine deamination in genomic DNA and cellular RNA. Here we report eight next-generation CBEs (BE4 with either RrA3F [wt, F130L], AmAPOBEC1, SsAPOBEC3B [wt, R54Q], or PpAPOBEC1 [wt, H122A, R33A]) that display comparable DNA on-target editing frequencies, whilst eliciting a 12- to 69-fold reduction in C-to-U edits in the transcriptome, and up to a 45-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Further, no enrichment of genome-wide C•G to T•A edits are observed in mammalian cells following transfection of mRNA encoding five of these next-generation editors. Taken together, these next-generation CBEs represent a collection of base editing tools for applications in which minimized off-target and high on-target activity are required., Cytosine base editors have been reported to induce off-target mutations in DNA and RNA. Here the authors identify next-generation CBEs with reduced guide-independent off-target editing profiles and retain high on-target editing activity.

/introductory paragraphCytosine base editors (CBEs) are molecular machines which enable efficient and programmable reversion of T•A to C•G point mutations in the human genome without induction of DNA double strand breaks1, 2. Recently, the foundational cytosine base editor (CBE) ‘BE3’, containing rAPOBEC1, was reported to induce unguided, genomic DNA3, 4 and cellular RNA5 cytosine deamination when expressed in living cells. To mitigate spurious off-target events, we developed a sensitive, high-throughput cellular assay to select next-generation CBEs that display reduced spurious deamination profiles relative to rAPOBEC1-based CBEs, whilst maintaining equivalent or superior on-target editing frequencies. We screened 153 CBEs containing cytidine deaminase enzymes with diverse sequences and identified four novel CBEs with the most promising on/off target ratios. These spurious-deamination-minimized CBEs (BE4 with either RrA3F, AmAPOBEC1, SsAPOBEC3B, or PpAPOBEC1) were further optimized for superior on- and off-target DNA editing profiles through structure-guided mutagenesis of the deaminase domain. These next-generation CBEs display comparable overall DNA on-target editing frequencies, whilst eliciting a 10- to 49-fold reduction in C-to-U edits in the transcriptome of treated cells, and up to a 33-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Taken together, these next-generation CBEs represent a new collection of base editing tools for applications in which minimization of spurious deamination is desirable and high on-target activity is required.

Osteoarthritis (OA), the most common form of arthritis, is a multi-factorial disease that primarily affects cartilage as well as other joint tissues such as subchondral bone. The lack of effective drug delivery, due to the avascular nature of cartilage and the rapid clearance of intra-articularly delivered drugs via the synovium, remains a major challenge in the development of disease mod- ifying drugs for OA. Cationic delivery carriers can significantly enhance the uptake, penetration and retention of drugs in cartilage by interacting with negatively charged matrix proteoglycans. In this study, we used “supercharged” green fluorescent proteins (GFPs), engineered to have a wide range of net positive charge and surface charge distributions, to characterize the effects of carrier charge on transport into cartilage in isolation of other factors such as carrier size and shape. We quantified the uptake, extent of cartilage penetration and cellular uptake of the GFP variants into living human knee cartilage and bovine cartilage explants. Based on these results, we identified optimal net charges of GFP carriers for potential drug targets located within cartilage extracellular matrix as well as the resident live chondrocytes. These cationic GFPs did not have adverse effects on cartilage in terms of measured cell viability and metabolism, cartilage cell biosynthesis and matrix degradation at doses needed for drug delivery. In addition to quantifying the kinetics of GFP uptake, we developed a predictive mathematical model for transport of the GFP variants that exhibited the highest uptake and penetration into cartilage. This model was further used to predict the transport behavior of GFPs during scale-up to in vivo applications such as intra-articular injection into human knees. The insights gained from this study set the stage for development of cartilage-targeted delivery systems to prevent cartilage degeneration, improve tissue regeneration and reduce inflammation that may cause degradation of other joint tissues affected by OA.

RNA-guided programmable nucleases from CRISPR systems generate precise breaks in DNA or RNA at specified positions. In cells, this activity can lead to changes in DNA sequence or RNA transcript abundance. Base editing is a newer genome editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks (DSBs). DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing byproducts. In this Review, we summarize base editing strategies to generate specific and precise point mutations in genomic DNA and RNA, highlight recent developments that expand the scope, specificity, precision, and in vivo delivery of base editors, and discuss limitations and future directions of base editing for research and therapeutic applications.

Programmable nucleases can introduce precise changes to genomic DNA through homology-directed repair (HDR). Unfortunately, HDR is largely restricted to mitotic cells, and is typically accompanied by an excess of stochastic insertions and deletions (indels). Here we present an in vivo base editing strategy that addresses these limitations. We use nuclease-free base editing to install a S33F mutation in β-catenin that blocks β-catenin phosphorylation, impedes β-catenin degradation, and upregulates Wnt signaling. In vitro, base editing installs the S33F mutation with a 200-fold higher editing:indel ratio than HDR. In post-mitotic cells in mouse inner ear, injection of base editor protein:RNA:lipid installs this mutation, resulting in Wnt activation that induces mitosis of cochlear supporting cells and cellular reprogramming. In contrast, injection of HDR agents does not induce Wnt upregulation. These results establish a strategy for modifying posttranslational states in signaling pathways, and an approach to precision editing in post-mitotic tissues., Base editing allows the precise introduction of point mutations into cellular DNA without requiring double-stranded DNA breaks or homology-directed repair, which is inefficient in postmitotic cells. Here the authors demonstrate in vivo base editing of post-mitotic somatic cells in the postnatal mouse inner ear with physiological outcomes.

A key limitation of the use of the CRISPR-Cas9 system for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), the required PAM sequence is NGG. No natural or engineered Cas9 variants that have been shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we use phage-assisted continuous evolution to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA and GAT. The PAM compatibility of xCas9 is the broadest reported, to our knowledge, among Cas9 proteins that are active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and cytidine and adenine base editing. Notably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility and DNA specificity.

Genome editing technologies are rapidly evolving, and analysis of deep sequencing data from target or off-target regions is necessary for measuring editing efficiency and evaluating safety. However, no software exists to analyze base editors, perform allele-specific quantification or that incorporates biologically-informed and scalable alignment approaches. Here, we present CRISPResso2 to fill this gap and illustrate its functionality by experimentally measuring and analyzing the editing properties of six genome editing agents.

SUMMARY The splicing factor SRSF1 promotes nonsense-mediated mRNA decay (NMD), a quality control mechanism that degrades mRNAs with premature termination codons (PTCs). Here we show that transcript-bound SRSF1 increases the binding of NMD factor UPF1 to mRNAs while in, or associated with, the nucleus, bypassing UPF2 recruitment and promoting NMD. SRSF1 promotes NMD when positioned downstream of a PTC, which resembles the mode of action of exon junction complex (EJC) and NMD factors. Moreover, splicing and/or EJC deposition increase the effect of SRSF1 on NMD. Lastly, SRSF1 enhances NMD of PTC-containing endogenous transcripts that result from various events. Our findings reveal an alternative mechanism for UPF1 recruitment, uncovering an additional connection between splicing and NMD. SRSF1’s role in the mRNA’s journey from splicing to decay has broad implications for gene expression regulation and genetic diseases., Graphical abstract In Brief: Aznarez et al. describe the mechanism behind the enhancement of nonsense-mediated mRNA decay (NMD) by the splicing factor SRSF1. Through its early association with the mRNA in the nucleus and its direct recruitment of UPF1, SRSF1 bypasses some of the steps necessary for the canonical NMD pathway.

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

Here we perform phage-assisted continuous evolution (PACE) of TEV protease, which canonically cleaves ENLYFQS, to cleave a very different target sequence, HPLVGHM, that is present in human IL-23. A protease emerging from ∼2500 generations of PACE contains 20 non-silent mutations, cleaves human IL-23 at the target peptide bond, and when pre-mixed with IL-23 in primary cultures of murine splenocytes inhibits IL-23-mediated immune signaling. We characterize the substrate specificity of this evolved enzyme, revealing shifted and broadened specificity changes at the six positions in which the target amino acid sequence differed. Mutational dissection and additional protease specificity profiling reveal the molecular basis of some of these changes. This work establishes the capability of changing the substrate specificity of a protease at many positions in a practical time scale and provides a foundation for the development of custom proteases that catalytically alter or destroy target proteins for biotechnological and therapeutic applications., Proteases are promising therapeutics to treat diseases such as hemophilia which are due to endogenous protease deficiency. Here the authors use phage-assisted continuous evolution to evolve a variant TEV protease with altered target peptide sequence specificities.

The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5-A7 and ~3.2× higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34

We report cellular RNA editing by adenine base editors and new ABE variants with reduced RNA editing activity., Adenine base editors (ABEs) enable precise and efficient conversion of target A•T base pairs to G•C base pairs in genomic DNA with a minimum of by-products. While ABEs have been reported to exhibit minimal off-target DNA editing, off-target editing of cellular RNA by ABEs has not been examined in depth. Here, we demonstrate that a current ABE generates low but detectable levels of widespread adenosine-to-inosine editing in cellular RNAs. Using structure-guided principles to design mutations in both deaminase domains, we developed new ABE variants that retain their ability to edit DNA efficiently but show greatly reduced RNA editing activity, as well as lower off-target DNA editing activity and reduced indel by-product formation, in three mammalian cell lines. By decoupling DNA and RNA editing activities, these ABE variants increase the precision of adenine base editing by minimizing both RNA and DNA off-target editing activity.

In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. In the presence of homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency and a low ratio of HDR products:indels. Here, we develop hRad51 mutants fused to Cas9(D10A) nickase (RDN) that mediate HDR while minimizing indels. We use RDN to install disease-associated point mutations in HEK293T cells with comparable or better efficiency than Cas9 nuclease and a 2.7-to-53-fold higher ratio of desired HDR product:undesired byproducts. Across five different human cell types, RDN variants generally result in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease, although HDR efficiencies remain strongly site- and cell type-dependent. RDN variants provide precision editing options in cell types amenable to HDR, especially when byproducts of DSBs must be minimized., Here the authors fuse hRad51 and variants thereof to Cas9 nickase to facilitate homology-directed repair without generating double strand breaks, minimizing indel formation and off-target editing. This tool represents progress towards the goal of performing HDR without an excess of undesired side products.

Genome editing technologies are rapidly evolving, and analysis of deep sequencing data from target or off-target regions is necessary for measuring editing efficiency and evaluating safety. However, no software exists to analyze base editors, perform allele-specific quantification or that incorporates biologically-informed and scalable alignment approaches. Here, we present CRISPResso2 to fill this gap and illustrate its functionality by experimentally measuring and analyzing the editing properties of six genome editing agents.

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.

The originally published article contained errors in reference numbering throughout table 1 (DNA base editors and their approximate editing windows) due to the unintended propagation of reference numbering from an earlier version of the table. The article has now been corrected online. The editors apologize for this error.

In this Article, owing to an error during the production process, in Fig. 1a, the dark blue and light blue wedges were incorrectly labelled as ‘G•C → T•A’ and ‘G•C → A•T’, instead of ‘C•G → T•A’ and ‘C•G → A•T’, respectively. Fig. 1 has been corrected online.

Proteins assemble into complexes with diverse quaternary structures. Although most heteromeric complexes of known structure have even stoichiometry, a significant minority have uneven stoichiometry-that is, differing numbers of each subunit type. To adopt this uneven stoichiometry, sequence-identical subunits must be asymmetric with respect to each other, forming different interactions within the complex. Here we first investigate the occurrence of uneven stoichiometry, demonstrating that it is common in vitro and is likely to be common in vivo. Next, we elucidate the structural determinants of uneven stoichiometry, identifying six different mechanisms by which it can be achieved. Finally, we study the frequency of uneven stoichiometry across evolution, observing a significant enrichment in bacteria compared with eukaryotes. We show that this arises due to a general increased tendency for bacterial proteins to self-assemble and form homomeric interactions, even within the context of a heteromeric complex.

Accessible summary • This paper will inform mental health service users and carers on how a University in Wales established a service user and carer-led research group. • The group's primary aim will be to undertake its own service user and carer-led research projects. • Mental health service users have undergone empowerment and research training at a University in Wales. This is an important initiative because it is the first service user and carer-led research group in Wales. • This paper is co-authored by a mental health service user and includes transcripts of service users’ stories written in their words. Abstract Service user and carer involvement in research has been gaining momentum in recent years. However, this involvement to date has primarily been as research respondents or ‘subjects’ in research studies. A group of mental health service users at a University in Wales underwent empowerment and research training to enable them to become active participants in the research process; this training was a necessary step to equip mental health service users with the skills to become independent researchers and to carry out service user-led research. We included transcripts from mental health service users on their views of the empowerment and research training received. We are not reporting, in this paper, on the findings from a research study rather it aims to inform readers how a service user and carer-led research group has been established in Wales. The group has two purposes: (1) to train service users in research methodologies, and thus for them to gain essential research skills; and (2) to undertake their own service user and carer-led research projects thereby implementing the research skills they have acquired from the training. The latter is a primary aim of the group; a future paper will report on its development.