Prediction of efficiencies for diverse prime editing systems in multiple cell types.

Applications of prime editing are often limited due to insufficient efficiencies, and it can require substantial time and resources to determine the most efficient pegRNAs and prime editors (PEs) to generate a desired edit under various experimental conditions. Here, we evaluated prime editing efficiencies for a total of 338,996 pairs of pegRNAs including 3,979 epegRNAs and target sequences in an error-free manner. These datasets enabled a systematic determination of factors affecting prime editing efficiencies. Then, we developed computational models, named DeepPrime and DeepPrime-FT, that can predict prime editing efficiencies for eight prime editing systems in seven cell types for all possible types of editing of up to 3 base pairs. We also extensively profiled the prime editing efficiencies at mismatched targets and developed a computational model predicting editing efficiencies at such targets. These computational models, together with our improved knowledge about prime editing efficiency determinants, will greatly facilitate prime editing applications.

Recording of elapsed time and temporal information about biological events using Cas9.

DNA has not been utilized to record temporal information, although DNA has been used to record biological information and to compute mathematical problems. Here, we found that indel generation by Cas9 and guide RNA can occur at steady rates, in contrast to typical dynamic biological reactions, and the accumulated indel frequency can be a function of time. By measuring indel frequencies, we developed a method for recording and measuring absolute time periods over hours to weeks in mammalian cells. These time-recordings were conducted in several cell types, with different promoters and delivery vectors for Cas9, and in both cultured cells and cells of living mice. As applications, we recorded the duration of chemical exposure and the lengths of elapsed time since the onset of biological events (e.g., heat exposure and inflammation). We propose that our systems could serve as synthetic "DNA clocks."

Massively parallel evaluation and computational prediction of the activities and specificities of 17 small Cas9s.

Recently, various small Cas9 orthologs and variants have been reported for use in in vivo delivery applications. Although small Cas9s are particularly suited for this purpose, selecting the most optimal small Cas9 for use at a specific target sequence continues to be challenging. Here, to this end, we have systematically compared the activities of 17 small Cas9s for thousands of target sequences. For each small Cas9, we have characterized the protospacer adjacent motif and determined optimal single guide RNA expression formats and scaffold sequence. High-throughput comparative analyses revealed distinct high- and low-activity groups of small Cas9s. We also developed DeepSmallCas9, a set of computational models predicting the activities of the small Cas9s at matched and mismatched target sequences. Together, this analysis and these computational models provide a useful guide for researchers to select the most suitable small Cas9 for specific applications.

SynDesign: web-based prime editing guide RNA design and evaluation tool for saturation genome editing.

Saturation genome editing (SGE) enables in-depth functional evaluation of disease-associated genes and variants by generating all possible single nucleotide variants (SNVs) within a given coding region. Although prime editing can be employed for inducing these SNVs, designing efficient prime editing guide RNAs (pegRNAs) can be challenging and time-consuming. Here, we present SynDesign, an easy-to-use webtool for the design, evaluation, and construction precision pegRNA libraries for SGE with synonymous mutation markers. SynDesign offers a simple yet powerful interface that automates the generation of all feasible pegRNA designs for a target gene or variant of interest. The pegRNAs are selected using the state-of-the-art models to predict prime editing efficiencies for various prime editors and cell types. Top-scoring pegRNA designs are further enhanced using synonymous mutation markers which improve pegRNA efficiency by diffusing the cellular mismatch repair mechanism and serve as sequence markers for improved identification of intended edits following deep sequencing. SynDesign is expected to facilitate future research using SGE to investigate genes or variants of interest associated with human diseases. SynDesign is freely available at https://deepcrispr.info/SynDesign without a login process.

Sniper2L is a high-fidelity Cas9 variant with high activity.

Although several high-fidelity SpCas9 variants have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences and developed DeepSniper, a deep learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.

In vivo adenine base editing rescues adrenoleukodystrophy in a humanized mouse model.

X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1, which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, very-long-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent ß-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fibroblasts at an efficiency of 7.4%. Adeno-associated virus (AAV)-mediated systemic delivery of NG-ABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction efficiency: ∼5.5%), spinal cord (∼5.1%), and adrenal gland (∼2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a significant step toward treating human ALD disease.

Magnetogenetics with Piezo1 Mechanosensitive Ion Channel for CRISPR Gene Editing.

Regulation of genetic activity in single cells and tissues is pivotal to determine key cellular functions in current biomedicine, yet the conventional biochemical activators lack spatiotemporal precision due to the diffusion-mediated slow kinetics and nonselectivity. Here, we describe a magnetogenetic method for target-specific activation of a clustered regularly interspaced short palindromic repeats (CRISPR) system for the regulation of intracellular proteins. We used magnetomechanical force generated by the magnetic nanostructure to activate pre-encoded Piezo1, the mechanosensitive ion channel, on the target cell. The activated Piezo1 further triggers the intracellular Ca2+ signaling pathway, inducing the pre-encoded genes to express genes of interest (GOIs), which is Cas9 protein for the CRISPR regulation of the target proteins. We demonstrated that this magnetogenetic CRISPR system successfully edits the target genome for both in vitro and pseudo-in vivo environments, providing a versatile magnetic platform for remote gene editing of animals with various size scales.

Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth.

KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.

Somatic Mutations in TSC1 and TSC2 Cause Focal Cortical Dysplasia.

Focal cortical dysplasia (FCD) is a major cause of the sporadic form of intractable focal epilepsies that require surgical treatment. It has recently been reported that brain somatic mutations in MTOR account for 15%-25% of FCD type II (FCDII), characterized by cortical dyslamination and dysmorphic neurons. However, the genetic etiologies of FCDII-affected individuals who lack the MTOR mutation remain unclear. Here, we performed deep hybrid capture and amplicon sequencing (read depth of 100×-20,012×) of five important mTOR pathway genes-PIK3CA, PIK3R2, AKT3, TSC1, and TSC2-by using paired brain and saliva samples from 40 FCDII individuals negative for MTOR mutations. We found that 5 of 40 individuals (12.5%) had brain somatic mutations in TSC1 (c.64C>T [p.Arg22Trp] and c.610C>T [p.Arg204Cys]) and TSC2 (c.4639G>A [p.Val1547Ile]), and these results were reproducible on two different sequencing platforms. All identified mutations induced hyperactivation of the mTOR pathway by disrupting the formation or function of the TSC1-TSC2 complex. Furthermore, in utero CRISPR-Cas9-mediated genome editing of Tsc1 or Tsc2 induced the development of spontaneous behavioral seizures, as well as cytomegalic neurons and cortical dyslamination. These results show that brain somatic mutations in TSC1 and TSC2 cause FCD and that in utero application of the CRISPR-Cas9 system is useful for generating neurodevelopmental disease models of somatic mutations in the brain.

Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption.

The use of paired Cas9 nickases instead of Cas9 nuclease drastically reduces off-target effects. Because both nickases must function for a nickase pair to make a double-strand break, the efficiency of paired nickases can intuitively be expected to be lower than that of either corresponding nuclease alone. Here, we carefully compared the gene-disrupting efficiency of Cas9 paired nickases with that of nucleases. Interestingly, the T7E1 assay and deep sequencing showed that on-target efficiency of paired D10A Cas9 nickases was frequently comparable, but sometimes higher than that of either corresponding nucleases in mammalian cells. As the underlying mechanism, we found that the HNH domain, which is preserved in the D10A Cas9 nickase, has higher activity than the RuvC domain in mammalian cells. In this study, we showed: (i) the in vivo cleavage efficiency of the HNH domain of Cas9 in mammalian cells is higher than that of the RuvC domain, (ii) paired Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. We envision that our findings which were overlooked in previous reports will serve as a new potential guideline for tool selection for CRISPR-Cas9-mediated gene disruption, facilitating efficient and precise genome editing.

In vivo gene correction with targeted sequence substitution through microhomology-mediated end joining.

Genome editing technology using programmable nucleases has rapidly evolved in recent years. The primary mechanism to achieve precise integration of a transgene is mainly based on homology-directed repair (HDR). However, an HDR-based genome-editing approach is less efficient than non-homologous end-joining (NHEJ). Recently, a microhomology-mediated end-joining (MMEJ)-based transgene integration approach was developed, showing feasibility both in vitro and in vivo. We expanded this method to achieve targeted sequence substitution (TSS) of mutated sequences with normal sequences using double-guide RNAs (gRNAs), and a donor template flanking the microhomologies and target sequence of the gRNAs in vitro and in vivo. Our method could realize more efficient sequence substitution than the HDR-based method in vitro using a reporter cell line, and led to the survival of a hereditary tyrosinemia mouse model in vivo. The proposed MMEJ-based TSS approach could provide a novel therapeutic strategy, in addition to HDR, to achieve gene correction from a mutated sequence to a normal sequence.

Concise Review: Fate Determination of Stem Cells by Deubiquitinating Enzymes.

Post-translational modification by ubiquitin molecules is a key regulatory process for stem cell fate determination. Ubiquitination and deubiquitination are the major cellular processes used to balance the protein turnover of several transcription factors that regulate stem cell differentiation. Deubiquitinating enzymes (DUBs), which facilitate the processing of ubiquitin, significantly influence stem cell fate choices. Specifically, DUBs play a critical regulatory role during development by directing the production of new specialized cells. This review focuses on the regulatory role of DUBs in various cellular processes, including stem cell pluripotency and differentiation, adult stem cell signaling, cellular reprogramming, spermatogenesis, and oogenesis. Specifically, the identification of interactions of DUBs with core transcription factors has provided new insight into the role of DUBs in regulating stem cell fate determination. Thus, DUBs have emerged as key pharmacologic targets in the search to develop highly specific agents to treat various illnesses. Stem Cells 2017;35:9-16.

In Situ Pluripotency Factor Expression Promotes Functional Recovery From Cerebral Ischemia.

Recovery from ischemic tissue injury can be promoted by cell proliferation and neovascularization. Transient expression of four pluripotency factors (Pou5f1, Sox2, Myc, and Klf4) has been used to convert cell types but never been tested as a means to promote functional recovery from ischemic injury. Here we aimed to determine whether transient in situ pluripotency factor expression can improve neurobehavioral function. Cerebral ischemia was induced by transient bilateral common carotid artery occlusion, after which the four pluripotency factors were expressed through either doxycycline administration into the lateral ventricle in transgenic mice in which the four factors are expressed in a doxycycline-inducible manner. Histologic evaluation showed that this transient expression induced the proliferative generation of astrocytes and/or neural progenitors, but not neurons or glial scar, and increased neovascularization with upregulation of angiogenic factors. Furthermore, in vivo pluripotency factor expression caused neuroprotective effects such as increased numbers of mature neurons and levels of synaptic markers in the striatum. Dysplasia or tumor development was not observed. Importantly, neurobehavioral evaluations such as rotarod and ladder walking tests showed that the expression of the four factors dramatically promoted functional restoration from ischemic injury. These results provide a basis for novel therapeutic modality development for cerebral ischemia.

Astroglial Activation by an Enriched Environment after Transplantation of Mesenchymal Stem Cells Enhances Angiogenesis after Hypoxic-Ischemic Brain Injury.

Transplantation of mesenchymal stem cells (MSCs) has paracrine effects; however, the effects are known to be largely limited. Here we investigated the combination effects of cell transplantation and enriched environment (EE) in a model of hypoxic-ischemic brain injury. Brain damage was induced in seven-day-old mice by unilateral carotid artery ligation and exposure to hypoxia (8% O2 for 90 min). At six weeks of age, the mice were randomly assigned to four groups: phosphate-buffered saline (PBS)-control (CON), PBS-EE, MSC-CON, and MSC-EE. Rotarod and grip strength tests were performed to evaluate neurobehavioral functions. Histologic evaluations were also performed to confirm the extent of astrocyte activation and endogenous angiogenesis. An array-based multiplex ELISA and Western blot were used to identify growth factors in vivo and in vitro. Two weeks after treatment, levels of astrocyte density and angiogenic factors were increased in MSC-EE mice, but glial scarring was not increased. Eight weeks after treatment, angiogenesis was increased, and behavioral outcomes were synergistically improved in the MSC-EE group. Astrocytes co-cultured with MSCs expressed higher levels of angiogenic factors than astrocytes cultured alone. The mechanisms of this synergistic effect included enhanced repair processes, such as increased endogenous angiogenesis and upregulation of angiogenic factors released from activated astrocytes.

Deep learning models to predict the editing efficiencies and outcomes of diverse base editors.

Applications of base editing are frequently restricted by the requirement for a protospacer adjacent motif (PAM), and selecting the optimal base editor (BE) and single-guide RNA pair (sgRNA) for a given target can be difficult. To select for BEs and sgRNAs without extensive experimental work, we systematically compared the editing windows, outcomes and preferred motifs for seven BEs, including two cytosine BEs, two adenine BEs and three C•G to G•C BEs at thousands of target sequences. We also evaluated nine Cas9 variants that recognize different PAM sequences and developed a deep learning model, DeepCas9variants, for predicting which variants function most efficiently at sites with a given target sequence. We then develop a computational model, DeepBE, that predicts editing efficiencies and outcomes of 63 BEs that were generated by incorporating nine Cas9 variants as nickase domains into the seven BE variants. The predicted median efficiencies of BEs with DeepBE-based design were 2.9- to 20-fold higher than those of rationally designed SpCas9-containing BEs.

In vivo application of base and prime editing to treat inherited retinal diseases.

Inherited retinal diseases (IRDs) are vision-threatening retinal disorders caused by pathogenic variants of genes related to visual functions. Genomic analyses in patients with IRDs have revealed pathogenic variants which affect vision. However, treatment options for IRDs are limited to nutritional supplements regardless of genetic variants or gene-targeting approaches based on antisense oligonucleotides and adeno-associated virus vectors limited to targeting few genes. Genome editing, particularly that involving clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies, can correct pathogenic variants and provide additional treatment opportunities. Recently developed base and prime editing platforms based on CRISPR-Cas9 technologies are promising for therapeutic genome editing because they do not employ double-stranded breaks (DSBs), which are associated with P53 activation, large deletions, and chromosomal translocations. Instead, using attached deaminases and reverse transcriptases, base and prime editing efficiently induces specific base substitutions and intended genetic changes (substitutions, deletions, or insertions), respectively, without DSBs. In this review, we will discuss the recent in vivo application of CRISPR-Cas9 technologies, focusing on base and prime editing, in animal models of IRDs.

DNA double-strand break-free CRISPR interference delays Huntington's disease progression in mice.

Huntington's disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. CRISPR-Cas9 nuclease causes double-strand breaks (DSBs) in the targeted DNA that induces toxicity, whereas CRISPR interference (CRISPRi) using dead Cas9 (dCas9) suppresses the target gene expression without DSBs. Delivery of dCas9-sgRNA targeting CAG repeat region does not damage the targeted DNA in HEK293T cells containing CAG repeats. When this study investigates whether CRISPRi can suppress mutant HTT (mHTT), CRISPRi results in reduced expression of mHTT with relative preservation of the wild-type HTT in human HD fibroblasts. Although both dCas9 and Cas9 treatments reduce mHTT by sgRNA targeting the CAG repeat region, CRISPRi delays behavioral deterioration and protects striatal neurons against cell death in HD mice. Collectively, CRISPRi can delay disease progression by suppressing mHtt, suggesting DNA DSB-free CRISPRi is a potential therapy for HD that can compensate for the shortcoming of CRISPR-Cas9 nuclease.

Basic Principles and Clinical Applications of CRISPR-Based Genome Editing.

Advances in sequencing technologies have facilitated the discovery of previously unknown genetic variants in both inherited and acquired disorders, and tools to correct these pathogenic variants are rapidly evolving. Since the first introduction of CRISPR-Cas9 in 2012, the field of CRISPR-based genome editing has progressed immensely, giving hope to many patients suffering from genetic disorders that lack effective treatment. In this review, we will examine the basic principles of CRISPR-based genome editing, explain the mechanisms of new genome editors, including base editors and prime editors, and evaluate the therapeutic possibilities of CRISPR-based genome editing by focusing on recently published clinical trials and animal studies. Although efficacy and safety issues remain a large concern, we cannot deny that CRISPR-based genome editing will soon be prevalent in clinical practice.

High-throughput functional evaluation of human cancer-associated mutations using base editors.

Comprehensive phenotypic characterization of the many mutations found in cancer tissues is one of the biggest challenges in cancer genomics. In this study, we evaluated the functional effects of 29,060 cancer-related transition mutations that result in protein variants on the survival and proliferation of non-tumorigenic lung cells using cytosine and adenine base editors and single guide RNA (sgRNA) libraries. By monitoring base editing efficiencies and outcomes using surrogate target sequences paired with sgRNA-encoding sequences on the lentiviral delivery construct, we identified sgRNAs that induced a single primary protein variant per sgRNA, enabling linking those mutations to the cellular phenotypes caused by base editing. The functions of the vast majority of the protein variants (28,458 variants, 98%) were classified as neutral or likely neutral; only 18 (0.06%) and 157 (0.5%) variants caused outgrowing and likely outgrowing phenotypes, respectively. We expect that our approach can be extended to more variants of unknown significance and other tumor types.

Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases.

The use of prime editing-a gene-editing technique that induces small genetic changes without the need for donor DNA and without causing double strand breaks-to correct pathogenic mutations and phenotypes needs to be tested in animal models of human genetic diseases. Here we report the use of prime editors 2 and 3, delivered by hydrodynamic injection, in mice with the genetic liver disease hereditary tyrosinemia, and of prime editor 2, delivered by an adeno-associated virus vector, in mice with the genetic eye disease Leber congenital amaurosis. For each pathogenic mutation, we identified an optimal prime-editing guide RNA by using cells transduced with lentiviral libraries of guide-RNA-encoding sequences paired with the corresponding target sequences. The prime editors precisely corrected the disease-causing mutations and led to the amelioration of the disease phenotypes in the mice, without detectable off-target edits. Prime editing should be tested further in more animal models of genetic diseases.