Publications by authors named "David R Liu"

200 Publications

The NIH Somatic Cell Genome Editing program.

Nature 2021 Apr 7;592(7853):195-204. Epub 2021 Apr 7.

Department of Neurosurgery, Yale University, New Haven, CT, USA.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.
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http://dx.doi.org/10.1038/s41586-021-03191-1DOI Listing
April 2021

Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.

Genome Biol 2021 Mar 16;22(1):83. Epub 2021 Mar 16.

Department of Medicine, Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, GA, 30912, USA.

Background: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter.

Results: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders.

Conclusions: PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.
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http://dx.doi.org/10.1186/s13059-021-02304-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962346PMC
March 2021

Massively parallel assessment of human variants with base editor screens.

Cell 2021 Feb;184(4):1064-1080.e20

Genetic Perturbation Platform, Broad Institute, Cambridge, MA 02142, USA. Electronic address:

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.
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http://dx.doi.org/10.1016/j.cell.2021.01.012DOI Listing
February 2021

Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity.

Science 2021 02;371(6531):803-810

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.

Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.
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http://dx.doi.org/10.1126/science.abf5972DOI Listing
February 2021

PrimeDesign software for rapid and simplified design of prime editing guide RNAs.

Nat Commun 2021 02 15;12(1):1034. Epub 2021 Feb 15.

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.

Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.
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http://dx.doi.org/10.1038/s41467-021-21337-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884779PMC
February 2021

Precision genome editing using cytosine and adenine base editors in mammalian cells.

Nat Protoc 2021 02 18;16(2):1089-1128. Epub 2021 Jan 18.

Merkin Institute of Transformative Technologies in Healthcare, The Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Genome editing has transformed the life sciences and has exciting prospects for use in treating genetic diseases. Our laboratory developed base editing to enable precise and efficient genome editing while minimizing undesired byproducts and toxicity associated with double-stranded DNA breaks. Adenine and cytosine base editors mediate targeted A•T-to-G•C or C•G-to-T•A base pair changes, respectively, which can theoretically address most human disease-associated single-nucleotide polymorphisms. Current base editors can achieve high editing efficiencies-for example, approaching 100% in cultured mammalian cells or 70% in adult mouse neurons in vivo. Since their initial description, a large set of base editor variants have been developed with different on-target and off-target editing characteristics. Here, we describe a protocol for using base editing in cultured mammalian cells. We provide guidelines for choosing target sites, appropriate base editor variants and delivery strategies to best suit a desired application. We further describe standard base-editing experiments in HEK293T cells, along with computational analysis of base-editing outcomes using CRISPResso2. Beginning with target DNA site selection, base-editing experiments in mammalian cells can typically be completed within 1-3 weeks and require only standard molecular biology techniques and readily available plasmid constructs.
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http://dx.doi.org/10.1038/s41596-020-00450-9DOI Listing
February 2021

Laboratory evolution of a sortase enzyme that modifies amyloid-β protein.

Nat Chem Biol 2021 03 11;17(3):317-325. Epub 2021 Jan 11.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Epitope-specific enzymes are powerful tools for site-specific protein modification but generally require genetic manipulation of the target protein. Here, we describe the laboratory evolution of the bacterial transpeptidase sortase A to recognize the LMVGG sequence in endogenous amyloid-β (Aβ) protein. Using a yeast display selection for covalent bond formation, we evolved a sortase variant that prefers LMVGG substrates from a starting enzyme that prefers LPESG substrates, resulting in a >1,400-fold change in substrate preference. We used this evolved sortase to label endogenous Aβ in human cerebrospinal fluid, enabling the detection of Aβ with sensitivities rivaling those of commercial assays. The evolved sortase can conjugate a hydrophilic peptide to Aβ, greatly impeding the ability of the resulting protein to aggregate into higher-order structures. These results demonstrate laboratory evolution of epitope-specific enzymes toward endogenous targets as a strategy for site-specific protein modification without target gene manipulation and enable potential future applications of sortase-mediated labeling of Aβ peptides.
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http://dx.doi.org/10.1038/s41589-020-00706-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904614PMC
March 2021

In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice.

Nature 2021 01 6;589(7843):608-614. Epub 2021 Jan 6.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Hutchinson-Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to approximately 14 years. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87-91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20-60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.
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http://dx.doi.org/10.1038/s41586-020-03086-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872200PMC
January 2021

Phage-assisted continuous and non-continuous evolution.

Nat Protoc 2020 12 16;15(12):4101-4127. Epub 2020 Nov 16.

Merkin Institute of Transformative Technologies in Healthcare, The Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Directed evolution, which applies the principles of Darwinian evolution to a laboratory setting, is a powerful strategy for generating biomolecules with diverse and tailored properties. This technique can be implemented in a highly efficient manner using continuous evolution, which enables the steps of directed evolution to proceed seamlessly over many successive generations with minimal researcher intervention. Phage-assisted continuous evolution (PACE) enables continuous directed evolution in bacteria by mapping the steps of Darwinian evolution onto the bacteriophage life cycle and allows directed evolution to occur on much faster timescales compared to conventional methods. This protocol provides detailed instructions on evolving proteins using PACE and phage-assisted non-continuous evolution (PANCE) and includes information on the preparation of selection phage and host cells, the assembly of a continuous flow apparatus and the performance and analysis of evolution experiments. This protocol can be performed in as little as 2 weeks to complete more than 100 rounds of evolution (complete cycles of mutation, selection and replication) in a single PACE experiment.
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http://dx.doi.org/10.1038/s41596-020-00410-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865204PMC
December 2020

Publisher Correction: Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing.

Nat Biomed Eng 2020 Nov;4(11):1119

Gavin Herbert Eye Institute, Department of Ophthalmology, University of California, Irvine, Irvine, CA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41551-020-00652-2DOI Listing
November 2020

Reactions to the National Academies/Royal Society Report on .

CRISPR J 2020 10;3(5):332-349

In September 2020, a detailed report on was published. The report offers a translational pathway for the limited approval of germline editing under limited circumstances and assuming various criteria have been met. In this perspective, some three dozen experts from the fields of genome editing, medicine, bioethics, law, and related fields offer their candid reactions to the National Academies/Royal Society report, highlighting areas of support, omissions, disagreements, and priorities moving forward.
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http://dx.doi.org/10.1089/crispr.2020.29106.manDOI Listing
October 2020

Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing.

Nat Biomed Eng 2021 02 19;5(2):169-178. Epub 2020 Oct 19.

Gavin Herbert Eye Institute, Department of Ophthalmology, University of California, Irvine, Irvine, CA, USA.

Cytosine base editors and adenine base editors (ABEs) can correct point mutations predictably and independent of Cas9-induced double-stranded DNA breaks (which causes substantial indel formation) and homology-directed repair (which typically leads to low editing efficiency). Here, we show, in adult mice, that a subretinal injection of a lentivirus expressing an ABE and a single-guide RNA targeting a de novo nonsense mutation in the Rpe65 gene corrects the pathogenic mutation with up to 29% efficiency and with minimal formation of indel and off-target mutations, despite the absence of the canonical NGG sequence as a protospacer-adjacent motif. The ABE-treated mice displayed restored RPE65 expression and retinoid isomerase activity, and near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.
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http://dx.doi.org/10.1038/s41551-020-00632-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885272PMC
February 2021

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

Science 2020 07;369(6503):566-571

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

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.
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http://dx.doi.org/10.1126/science.abb1390DOI Listing
July 2020

Multimodal small-molecule screening for human prion protein binders.

J Biol Chem 2020 09 28;295(39):13516-13531. Epub 2020 Jul 28.

Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA; Prion Alliance, Cambridge, Massachusetts, USA; Harvard Medical School, Boston, Massachusetts, USA. Electronic address:

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including F-observed and saturation transfer difference (STD) NMR spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak ( > 1 mm), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. Whereas orthogonal binder discovery methods could yield high-affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease.
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http://dx.doi.org/10.1074/jbc.RA120.014905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521658PMC
September 2020

A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.

Nature 2020 07 8;583(7817):631-637. Epub 2020 Jul 8.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques. Because previously described cytidine deaminases operate on single-stranded nucleic acids, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.
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http://dx.doi.org/10.1038/s41586-020-2477-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381381PMC
July 2020

Programmable mA modification of cellular RNAs with a Cas13-directed methyltransferase.

Nat Biotechnol 2020 12 29;38(12):1431-1440. Epub 2020 Jun 29.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

N-Methyladenosine (mA) is the most widespread internal messenger RNA modification in humans. Despite recent progress in understanding the biological roles of mA, the inability to install mA site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific mA and phenotypic outcomes. In the present study, we demonstrate that nucleus-localized dCas13 fusions with a truncated METTL3 methyltransferase domain and cytoplasm-localized fusions with a modified METTL3:METTL14 methyltransferase complex can direct site-specific mA incorporation in distinct cellular compartments, with the former fusion protein having particularly low off-target activity. Independent cellular assays across multiple sites confirm that this targeted RNA methylation (TRM) system mediates efficient mA installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce mA-mediated changes to transcript abundance and alternative splicing. These findings establish TRM as a tool for targeted epitranscriptome engineering that can reveal the effect of individual mA modifications and dissect their functional roles.
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http://dx.doi.org/10.1038/s41587-020-0572-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718427PMC
December 2020

Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.

Nat Biotechnol 2020 07 22;38(7):824-844. Epub 2020 Jun 22.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR-Cas-derived genome editing agents-nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.
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http://dx.doi.org/10.1038/s41587-020-0561-9DOI Listing
July 2020

Determinants of Base Editing Outcomes from Target Library Analysis and Machine Learning.

Cell 2020 07 12;182(2):463-480.e30. Epub 2020 Jun 12.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Although base editors are widely used to install targeted point mutations, the factors that determine base editing outcomes are not well understood. We characterized sequence-activity relationships of 11 cytosine and adenine base editors (CBEs and ABEs) on 38,538 genomically integrated targets in mammalian cells and used the resulting outcomes to train BE-Hive, a machine learning model that accurately predicts base editing genotypic outcomes (R ≈ 0.9) and efficiency (R ≈ 0.7). We corrected 3,388 disease-associated SNVs with ≥90% precision, including 675 alleles with bystander nucleotides that BE-Hive correctly predicted would not be edited. We discovered determinants of previously unpredictable C-to-G, or C-to-A editing and used these discoveries to correct coding sequences of 174 pathogenic transversion SNVs with ≥90% precision. Finally, we used insights from BE-Hive to engineer novel CBE variants that modulate editing outcomes. These discoveries illuminate base editing, enable editing at previously intractable targets, and provide new base editors with improved editing capabilities.
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http://dx.doi.org/10.1016/j.cell.2020.05.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384975PMC
July 2020

In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness.

Sci Transl Med 2020 06;12(546)

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.

Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide therapeutic restoration of gene function. Mutations of transmembrane channel-like 1 gene () can cause dominant or recessive deafness. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (; resulting in the substitution p.Y182C) in Tmc1 that causes deafness. encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. We found that sensory hair cells of Baringo mice have a complete loss of auditory sensory transduction. To repair the mutation, we tested several optimized cytosine base editors (CBEmax variants) and guide RNAs in Baringo mouse embryonic fibroblasts. We packaged the most promising CBE, derived from an activation-induced cytidine deaminase (AID), into dual adeno-associated viruses (AAVs) using a split-intein delivery system. The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Injected mice showed up to 51% reversion of the point mutation to wild-type sequence () in transcripts. Repair of in vivo restored inner hair cell sensory transduction and hair cell morphology and transiently rescued low-frequency hearing 4 weeks after injection. These findings provide a foundation for a potential one-time treatment for recessive hearing loss and support further development of base editing to correct pathogenic point mutations.
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http://dx.doi.org/10.1126/scitranslmed.aay9101DOI Listing
June 2020

The developing toolkit of continuous directed evolution.

Nat Chem Biol 2020 06 22;16(6):610-619. Epub 2020 May 22.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Continuous directed evolution methods allow the key steps of evolution-gene diversification, selection, and replication-to proceed in the laboratory with minimal researcher intervention. As a result, continuous evolution can find solutions much more quickly than traditional discrete evolution methods. Continuous evolution also enables the exploration of longer and more numerous evolutionary trajectories, increasing the likelihood of accessing solutions that require many steps through sequence space and greatly facilitating the iterative refinement of selection conditions and targeted mutagenesis strategies. Here we review the historical advances that have expanded continuous evolution from its earliest days as an experimental curiosity to its present state as a powerful and surprisingly general strategy for generating tailor-made biomolecules, and discuss more recent improvements with an eye to the future.
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http://dx.doi.org/10.1038/s41589-020-0532-yDOI Listing
June 2020

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

Nat Biotechnol 2020 Jul;38(7):901

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41587-020-0562-8DOI Listing
July 2020

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

Nat Biotechnol 2020 07 16;38(7):883-891. Epub 2020 Mar 16.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

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 (k) 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.
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http://dx.doi.org/10.1038/s41587-020-0453-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357821PMC
July 2020

Glucose Response by Stem Cell-Derived β Cells In Vitro Is Inhibited by a Bottleneck in Glycolysis.

Cell Rep 2020 05;31(6):107623

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA. Electronic address:

Stem cell-derived β (SC-β) cells could provide unlimited human β cells toward a curative diabetes treatment. Differentiation of SC-β cells yields transplantable islets that secrete insulin in response to glucose challenges. Following transplantation into mice, SC-β cell function is comparable to human islets, but the magnitude and consistency of response in vitro are less robust than observed in cadaveric islets. Here, we profile metabolism of SC-β cells and islets to quantify their capacity to sense glucose and identify reduced anaplerotic cycling in the mitochondria as the cause of reduced glucose-stimulated insulin secretion in SC-β cells. This activity can be rescued by challenging SC-β cells with intermediate metabolites from the TCA cycle and late but not early glycolysis, downstream of the enzymes glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. Bypassing this metabolic bottleneck results in a robust, bi-phasic insulin release in vitro that is identical in magnitude to functionally mature human islets.
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http://dx.doi.org/10.1016/j.celrep.2020.107623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7433758PMC
May 2020

Prime genome editing in rice and wheat.

Nat Biotechnol 2020 05 16;38(5):582-585. Epub 2020 Mar 16.

State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.

Prime editors, which are CRISPR-Cas9 nickase (H840A)-reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%.
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http://dx.doi.org/10.1038/s41587-020-0455-xDOI Listing
May 2020

Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope.

Nat Commun 2020 04 24;11(1):1979. Epub 2020 Apr 24.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, 01605, USA.

CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.
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http://dx.doi.org/10.1038/s41467-020-15892-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181807PMC
April 2020

Continuous evolution of SpCas9 variants compatible with non-G PAMs.

Nat Biotechnol 2020 04 10;38(4):471-481. Epub 2020 Feb 10.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

The targeting scope of Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants is largely restricted to protospacer-adjacent motif (PAM) sequences containing G bases. Here we report the evolution of three new SpCas9 variants that collectively recognize NRNH PAMs (where R is A or G and H is A, C or T) using phage-assisted non-continuous evolution, three new phage-assisted continuous evolution 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 mediated indel formation and base editing in human cells and enabled A•T-to-G•C base editing of a sickle cell anemia mutation using a previously inaccessible CACC PAM. These new evolved SpCas9 variants, together with previously reported variants, in principle enable targeting of most NR PAM sequences and substantially reduce the fraction of genomic sites that are inaccessible by Cas9-based methods.
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http://dx.doi.org/10.1038/s41587-020-0412-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145744PMC
April 2020

Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors.

Nat Biotechnol 2020 05 10;38(5):620-628. Epub 2020 Feb 10.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of genome-wide Cas9-independent off-target C•G-to-T•A mutation in mouse embryos and in rice. Here we develop multiple rapid, cost-effective methods to screen the propensity of different CBEs to induce Cas9-independent deamination in Escherichia coli and in human cells. We use these assays to identify CBEs with reduced Cas9-independent deamination and validate via whole-genome sequencing that YE1, a narrowed-window CBE variant, displays background levels of Cas9-independent off-target editing. We engineered YE1 variants that retain the substrate-targeting scope of high-activity CBEs while maintaining minimal Cas9-independent off-target editing. The suite of CBEs characterized and engineered in this study collectively offer ~10-100-fold lower average Cas9-independent off-target DNA editing while maintaining robust on-target editing at most positions targetable by canonical CBEs, and thus are especially promising for applications in which off-target editing must be minimized.
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http://dx.doi.org/10.1038/s41587-020-0414-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335424PMC
May 2020

Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses.

Nat Biomed Eng 2020 01 14;4(1):97-110. Epub 2020 Jan 14.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

The success of base editors for the study and treatment of genetic diseases depends on the ability to deliver them in vivo to the relevant cell types. Delivery via adeno-associated viruses (AAVs) is limited by AAV packaging capacity, which precludes the use of full-length base editors. Here, we report the application of dual AAVs for the delivery of split cytosine and adenine base editors that are then reconstituted by trans-splicing inteins. Optimized dual AAVs enable in vivo base editing at therapeutically relevant efficiencies and dosages in the mouse brain (up to 59% of unsorted cortical tissue), liver (38%), retina (38%), heart (20%) and skeletal muscle (9%). We also show that base editing corrects, in mouse brain tissue, a mutation that causes Niemann-Pick disease type C (a neurodegenerative ataxia), slowing down neurodegeneration and increasing lifespan. The optimized delivery vectors should facilitate the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest.
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http://dx.doi.org/10.1038/s41551-019-0501-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980783PMC
January 2020

High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells.

Nat Biomed Eng 2020 01 14;4(1):111-124. Epub 2020 Jan 14.

Department of Pharmacology, Yonsei University College of Medicine, Seoul, Republic of Korea.

The applications of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing can be limited by a lack of compatible protospacer adjacent motifs (PAMs), insufficient on-target activity and off-target effects. Here, we report an extensive comparison of the PAM-sequence compatibilities and the on-target and off-target activities of Cas9 from Streptococcus pyogenes (SpCas9) and the SpCas9 variants xCas9 and SpCas9-NG (which are known to have broader PAM compatibility than SpCas9) at 26,478 lentivirally integrated target sequences and 78 endogenous target sites in human cells. We found that xCas9 has the lowest tolerance for mismatched target sequences and that SpCas9-NG has the broadest PAM compatibility. We also show, on the basis of newly identified non-NGG PAM sequences, that SpCas9-NG and SpCas9 can edit six previously unedited endogenous sites associated with genetic diseases. Moreover, we provide deep-learning models that predict the activities of xCas9 and SpCas9-NG at the target sequences. The resulting deeper understanding of the activities of xCas9, SpCas9-NG and SpCas9 in human cells should facilitate their use.
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http://dx.doi.org/10.1038/s41551-019-0505-1DOI Listing
January 2020

Adenine base editing in an adult mouse model of tyrosinaemia.

Nat Biomed Eng 2020 01 25;4(1):125-130. Epub 2019 Feb 25.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.

In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.
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http://dx.doi.org/10.1038/s41551-019-0357-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986236PMC
January 2020