Publications by authors named "Luke W Koblan"

16 Publications

  • Page 1 of 1

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

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

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

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

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

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

Search-and-replace genome editing without double-strand breaks or donor DNA.

Nature 2019 12 21;576(7785):149-157. Epub 2019 Oct 21.

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

Most genetic variants that contribute to disease are challenging to correct efficiently and without excess byproducts. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.
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http://dx.doi.org/10.1038/s41586-019-1711-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6907074PMC
December 2019

Publisher Correction: Continuous evolution of base editors with expanded target compatibility and improved activity.

Nat Biotechnol 2019 Sep;37(9):1091

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/s41587-019-0253-5DOI Listing
September 2019

Continuous evolution of base editors with expanded target compatibility and improved activity.

Nat Biotechnol 2019 09 22;37(9):1070-1079. Epub 2019 Jul 22.

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

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.
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http://dx.doi.org/10.1038/s41587-019-0193-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728210PMC
September 2019

Addendum: High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases.

Nat Commun 2019 07 2;10(1):3003. Epub 2019 Jul 2.

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

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http://dx.doi.org/10.1038/s41467-019-10892-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606619PMC
July 2019

High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases.

Nat Commun 2019 04 26;10(1):1937. Epub 2019 Apr 26.

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

The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development.
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http://dx.doi.org/10.1038/s41467-019-09987-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486577PMC
April 2019

Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction.

Nat Biotechnol 2018 10 29;36(9):843-846. Epub 2018 May 29.

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

Base editors enable targeted single-nucleotide conversions in genomic DNA. Here we show that expression levels are a bottleneck in base-editing efficiency. We optimize cytidine (BE4) and adenine (ABE7.10) base editors by modification of nuclear localization signals (NLS) and codon usage, and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max, and ABEmax editors correct pathogenic SNPs with substantially increased efficiency in a variety of mammalian cell types.
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http://dx.doi.org/10.1038/nbt.4172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126947PMC
October 2018

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

Sci Adv 2017 08 30;3(8):eaao4774. Epub 2017 Aug 30.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.

We recently developed base editing, the programmable conversion of target C:G base pairs to T:A without inducing double-stranded DNA breaks (DSBs) or requiring homology-directed repair using engineered fusions of Cas9 variants and cytidine deaminases. Over the past year, the third-generation base editor (BE3) and related technologies have been successfully used by many researchers in a wide range of organisms. The product distribution of base editing-the frequency with which the target C:G is converted to mixtures of undesired by-products, along with the desired T:A product-varies in a target site-dependent manner. We characterize determinants of base editing outcomes in human cells and establish that the formation of undesired products is dependent on uracil N-glycosylase (UNG) and is more likely to occur at target sites containing only a single C within the base editing activity window. We engineered CDA1-BE3 and AID-BE3, which use cytidine deaminase homologs that increase base editing efficiency for some sequences. On the basis of these observations, we engineered fourth-generation base editors (BE4 and SaBE4) that increase the efficiency of C:G to T:A base editing by approximately 50%, while halving the frequency of undesired by-products compared to BE3. Fusing BE3, BE4, SaBE3, or SaBE4 to Gam, a bacteriophage Mu protein that binds DSBs greatly reduces indel formation during base editing, in most cases to below 1.5%, and further improves product purity. BE4, SaBE4, BE4-Gam, and SaBE4-Gam represent the state of the art in C:G-to-T:A base editing, and we recommend their use in future efforts.
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http://dx.doi.org/10.1126/sciadv.aao4774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576876PMC
August 2017

Assessment of Bromodomain Target Engagement by a Series of BI2536 Analogues with Miniaturized BET-BRET.

ChemMedChem 2016 Dec 15;11(23):2575-2581. Epub 2016 Nov 15.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.

Evaluating the engagement of a small molecule ligand with a protein target in cells provides useful information for chemical probe optimization and pharmaceutical development. While several techniques exist that can be performed in a low-throughput manner, systematic evaluation of large compound libraries remains a challenge. In-cell engagement measurements are especially useful when evaluating compound classes suspected to target multiple cellular factors. In this study we used a bioluminescent resonant energy transfer assay to assess bromodomain engagement by a compound series containing bromodomain- and kinase-biasing polypharmacophores based on the known dual BRD4 bromodomain/PLK1 kinase inhibitor BI2536. With this assay, we discovered several novel agents with bromodomain-selective specificity profiles and cellular activity. Thus, this platform aids in distinguishing molecules whose cellular activity is difficult to assess due to polypharmacologic effects.
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http://dx.doi.org/10.1002/cmdc.201600502DOI Listing
December 2016

A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity.

Nat Chem Biol 2014 Aug 6;10(8):656-63. Epub 2014 Jul 6.

1] The Eli and Edythe L. Broad Institute, Cambridge, Massachusetts, USA. [2] Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. [3] Harvard Medical School, Boston, Massachusetts, USA. [4] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.

The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid- and nitrile-containing compounds provided structure-activity relationships in both potency and selectivity dimensions from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling and suggest that such profiling can be incorporated into the earliest stages of drug discovery.
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http://dx.doi.org/10.1038/nchembio.1578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5953424PMC
August 2014