Publications by authors named "Benjamin P Kleinstiver"

38 Publications

Enhanced Homology-directed Repair for Highly Efficient Gene Editing in Hematopoietic Stem/Progenitor Cells.

Blood 2021 Feb 23. Epub 2021 Feb 23.

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States.

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using SpCas9 and an oligodeoxynucleotide donor to repair genetic mutations demonstrated the capability to restore physiological protein expression, but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we show transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology directed repair (HDR) to achieve highly efficient (80% gp91phox+ cells compared to healthy donor control) long-term correction of X-CGD CD34+ cells.
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http://dx.doi.org/10.1182/blood.2020008503DOI Listing
February 2021

Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA.

Nat Protoc 2021 03 5;16(3):1511-1547. Epub 2021 Feb 5.

Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.

The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR-Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR-Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks.
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http://dx.doi.org/10.1038/s41596-020-00465-2DOI Listing
March 2021

Plant genome editing branches out.

Nat Plants 2021 01;7(1):4-5

Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.

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http://dx.doi.org/10.1038/s41477-020-00840-7DOI Listing
January 2021

Cell-based artificial APC resistant to lentiviral transduction for efficient generation of CAR-T cells from various cell sources.

J Immunother Cancer 2020 09;8(2)

Cellular Immunotherapy Program, Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA

Background: Adoptive cell therapy with chimeric antigen receptor T cells (CAR-T) has become a standard treatment for patients with certain aggressive B cell malignancies and holds promise to improve the care of patients suffering from numerous other cancers in the future. However, the high manufacturing cost of CAR-T cell therapies poses a major barrier to their broader clinical application. Among the key cost drivers of CAR-T production are single-use reagents for T cell activation and clinical-grade viral vector. The presence of variable amounts of contaminating monocytes in the starting material poses an additional challenge to CAR-T manufacturing, since they can impede T cell stimulation and transduction, resulting in manufacturing failure.

Methods: We created K562-based artificial antigen-presenting cells (aAPC) with genetically encoded T cell stimulation and costimulation that represent an inexhaustible source for T cell activation. We additionally disrupted endogenous expression of the low-density lipoprotein receptor (LDLR) on these aAPC (aAPC-ΔLDLR) using CRISPR-Cas9 gene editing nucleases to prevent inadvertent lentiviral transduction and avoid the sink effect on viral vector during transduction. Using various T cell sources, we produced CD19-directed CAR-T cells via aAPC-ΔLDLR-based activation and tested their in vitro and in vivo antitumor potency against B cell malignancies.

Results: We found that lack of LDLR expression on our aAPC-ΔLDLR conferred resistance to lentiviral transduction during CAR-T production. Using aAPC-ΔLDLR, we achieved efficient expansion of CAR-T cells even from unpurified starting material like peripheral blood mononuclear cells or unmanipulated leukapheresis product, containing substantial proportions of monocytes. CD19-directed CAR-T cells that we produced via aAPC-ΔLDLR-based expansion demonstrated potent antitumor responses in preclinical models of acute lymphoblastic leukemia and B-cell lymphoma.

Conclusions: Our aAPC-ΔLDLR represent an attractive approach for manufacturing of lentivirally transduced T cells that may be simpler and more cost efficient than currently available methods.
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http://dx.doi.org/10.1136/jitc-2020-000990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477986PMC
September 2020

Optimization of AsCas12a for combinatorial genetic screens in human cells.

Nat Biotechnol 2021 01 13;39(1):94-104. Epub 2020 Jul 13.

Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.
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http://dx.doi.org/10.1038/s41587-020-0600-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854777PMC
January 2021

Publisher Correction: Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.

Nat Biotechnol 2020 Jul;38(7):901

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, 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/s41587-020-0587-zDOI Listing
July 2020

Mutant Allele-Specific CRISPR Disruption in DYT1 Dystonia Fibroblasts Restores Cell Function.

Mol Ther Nucleic Acids 2020 Sep 15;21:1-12. Epub 2020 May 15.

Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Electronic address:

Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302-332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function.
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http://dx.doi.org/10.1016/j.omtn.2020.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270506PMC
September 2020

Author Correction: Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer.

Nat Microbiol 2020 Jun;5(6):872

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, 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/s41564-020-0726-9DOI Listing
June 2020

Listeria Phages Induce Cas9 Degradation to Protect Lysogenic Genomes.

Cell Host Microbe 2020 07 22;28(1):31-40.e9. Epub 2020 Apr 22.

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Innovative Genomics Institute, Berkeley, CA 94720, USA. Electronic address:

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy phage (viral) DNA. Phages, in turn, have evolved diverse "anti-CRISPR" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode two to three distinct anti-Cas9 proteins, with acrIIA1 always present. However, the significance of AcrIIA1's pervasiveness and its mechanism are unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain. During lysogeny in Listeria, AcrIIA1 triggers Cas9 degradation. During lytic infection, however, AcrIIA1 fails to block Cas9 due to its multi-step inactivation mechanism. Thus, phages encode an additional Acr that rapidly binds and inactivates Cas9. AcrIIA1 also uniquely inhibits a highly diverged Cas9 found in Listeria (similar to SauCas9) and Type II-C Cas9s, likely due to Cas9 HNH domain conservation. In summary, Listeria phages inactivate Cas9 in lytic growth using variable, narrow-spectrum inhibitors, while the broad-spectrum AcrIIA1 stimulates Cas9 degradation for protection of the lysogenic genome.
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http://dx.doi.org/10.1016/j.chom.2020.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351598PMC
July 2020

Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer.

Nat Microbiol 2020 04 26;5(4):620-629. Epub 2020 Mar 26.

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.

CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16-19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9-Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR-Cas9 inhibition.
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http://dx.doi.org/10.1038/s41564-020-0692-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194981PMC
April 2020

Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.

Science 2020 04 26;368(6488):290-296. Epub 2020 Mar 26.

Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.
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http://dx.doi.org/10.1126/science.aba8853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297043PMC
April 2020

In vivo engineering of lymphocytes after systemic exosome-associated AAV delivery.

Sci Rep 2020 03 11;10(1):4544. Epub 2020 Mar 11.

Department of Neurology, Massachusetts General Hospital, Charlestown, MA, USA.

Ex-vivo gene therapy using stem cells or T cells transduced by retroviral or lentiviral vectors has shown remarkable efficacy in the treatment of immunodeficiencies and cancer. However, the process is expensive, technically challenging, and not readily scalable to large patient populations, particularly in underdeveloped parts of the world. Direct in vivo gene therapy would avoid these issues, and such approaches with adeno-associated virus (AAV) vectors have been shown to be safe and efficacious in clinical trials for diseases affecting differentiated tissues such as the liver and CNS. However, the ability to transduce lymphocytes with AAV in vivo after systemic delivery has not been carefully explored. Here, we show that both standard and exosome-associated preparations of AAV8 vectors can effectively transduce a variety of immune cell populations including CD4 T cells, CD8 T cells, B cells, macrophages, and dendritic cells after systemic delivery in mice. We provide direct evidence of T cell transduction through the detection of AAV genomes and transgene mRNA, and show that intracellular and transmembrane proteins can be expressed. These findings establish the feasibility of AAV-mediated in vivo gene delivery to immune cells which will facilitate both basic and applied research towards the goal of direct in vivo gene immunotherapies.
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http://dx.doi.org/10.1038/s41598-020-61518-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066196PMC
March 2020

High levels of AAV vector integration into CRISPR-induced DNA breaks.

Nat Commun 2019 09 30;10(1):4439. Epub 2019 Sep 30.

Department of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA.

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.
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http://dx.doi.org/10.1038/s41467-019-12449-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769011PMC
September 2019

Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss.

Nat Med 2019 07 3;25(7):1123-1130. Epub 2019 Jul 3.

Department of Neurobiology, Harvard Medical School, Boston, MA, USA.

Since most dominant human mutations are single nucleotide substitutions, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve because commonly used endonucleases, such as Streptococcus pyogenes Cas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethoven mice, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells. We identified a PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.
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http://dx.doi.org/10.1038/s41591-019-0500-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802276PMC
July 2019

Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.

Nat Biotechnol 2019 03 11;37(3):276-282. Epub 2019 Feb 11.

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

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM). To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.
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http://dx.doi.org/10.1038/s41587-018-0011-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401248PMC
March 2019

Discovery of widespread type I and type V CRISPR-Cas inhibitors.

Science 2018 10 6;362(6411):240-242. Epub 2018 Sep 6.

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.

Bacterial CRISPR-Cas systems protect their host from bacteriophages and other mobile genetic elements. Mobile elements, in turn, encode various anti-CRISPR (Acr) proteins to inhibit the immune function of CRISPR-Cas. To date, Acr proteins have been discovered for type I (subtypes I-D, I-E, and I-F) and type II (II-A and II-C) but not other CRISPR systems. Here, we report the discovery of 12 genes, including inhibitors of type V-A and I-C CRISPR systems. AcrVA1 inhibits a broad spectrum of Cas12a (Cpf1) orthologs-including MbCas12a, Mb3Cas12a, AsCas12a, and LbCas12a-when assayed in human cells. The genes reported here provide useful biotechnological tools and mark the discovery of loci in many bacteria and phages.
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http://dx.doi.org/10.1126/science.aau5174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520112PMC
October 2018

Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs.

Nat Biomed Eng 2018 Jan 10;2(1):38-47. Epub 2018 Jan 10.

Microsoft Research, Cambridge, MA, USA.

The CRISPR-Cas9 system provides unprecedented genome editing capabilities. However, off-target effects lead to sub-optimal usage and additionally are a bottleneck in the development of therapeutic uses. Herein, we introduce the first machine learning-based approach to off-target prediction, yielding a state-of-the-art model for CRISPR-Cas9 that outperforms all other guide design services. Our approach, Elevation, consists of two interdependent machine learning models-one for scoring individual guide-target pairs, and another which aggregates these guide-target scores into a single, overall summary guide score. Through systematic investigation, we demonstrate that Elevation performs substantially better than competing approaches on both tasks. Additionally, we are the first to systematically evaluate approaches on the guide summary score problem; we show that the most widely-used method performs no better than random at times, whereas Elevation consistently outperformed it, sometimes by an order of magnitude. We also introduce an evaluation method that balances errors between active and inactive guides, thereby encapsulating a range of practical use cases; Elevation is consistently superior to other methods across the entire range. Finally, because of the large scale and computational demands of off-target prediction, we have developed a cloud-based service for quick retrieval. This service provides end-to-end guide design by also incorporating our previously reported on-target model, Azimuth. (https://crispr.ml:please treat this web site as confidential until publication).
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http://dx.doi.org/10.1038/s41551-017-0178-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6037314PMC
January 2018

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize.

Plant Biotechnol J 2019 02 22;17(2):362-372. Epub 2018 Jul 22.

Crop Bioengineering Center, Iowa State University, Ames, IA, USA.

CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9-guide RNA (gRNA) and LbCas12a-CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium-mediated transformation. On-target mutation analysis showed that 90%-100% of the Cas9-edited T0 plants carried indel mutations and 63%-77% of them were homozygous or biallelic mutants. In contrast, 0%-60% of Cas12a-edited T0 plants had on-target mutations. We then conducted CIRCLE-seq analysis to identify genome-wide potential off-target sites for Cas9. A total of 18 and 67 potential off-targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off-target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.
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http://dx.doi.org/10.1111/pbi.12982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320322PMC
February 2019

CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer's Disease.

Mol Ther Nucleic Acids 2018 Jun 16;11:429-440. Epub 2018 Mar 16.

Departments of Neurology and Radiology, Massachusetts General Hospital and Center for NeuroDiscovery, Harvard Medical School, Boston, MA, USA; Department of Public Health and Caring Sciences, Geriatrics, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden. Electronic address:

The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer's disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APP allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APP or APP. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APP-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APP transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APP allele both ex vivo and in vivo-and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.
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http://dx.doi.org/10.1016/j.omtn.2018.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992788PMC
June 2018

Temporal and Spatial Post-Transcriptional Regulation of Zebrafish mRNA by Long Noncoding RNA During Brain Vascular Assembly.

Arterioscler Thromb Vasc Biol 2018 07 3;38(7):1562-1575. Epub 2018 May 3.

From the Division of Neonatology, Department of Pediatrics (C.K., K.L., S.P., R.R.)

Objective: Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell-specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of mRNA expression is not understood, but it might partly explain Tie1's differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the locus- (), which regulates mRNA levels in zebrafish-we attempted to identify the mechanism of this regulation.

Approach And Results: Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that long noncoding RNA interacts with an RNA binding protein-embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)-that regulates mRNA levels. When we disrupted the interaction between and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)-mediated CRISPRi (CRISPR-mediated interference) to knock down . Treatment of the morpholino oligonucleotide-injected embryos with a small molecule that decreased mRNA levels rescued all 3 abnormal phenotypes.

Conclusions: We identified a novel mode of temporal and spatial post-transcriptional regulation of mRNA. It involves long noncoding RNA, , and Elavl1 (an interactor of ).
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http://dx.doi.org/10.1161/ATVBAHA.118.310848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023729PMC
July 2018

Allele-Specific CRISPR-Cas9 Genome Editing of the Single-Base P23H Mutation for Rhodopsin-Associated Dominant Retinitis Pigmentosa.

CRISPR J 2018 02;1:55-64

1 Ocular Genomics Institute , Massachusetts Eye and Ear Infirmary, Boston, Massachusetts.

Treatment strategies for dominantly inherited disorders typically involve silencing or ablating the pathogenic allele. CRISPR-Cas nucleases have shown promise in allele-specific knockout approaches when the dominant allele creates unique protospacer adjacent motifs that can lead to allele-restricted targeting. Here, we present a spacer-mediated allele-specific knockout approach that utilizes both SpCas9 variants and truncated single-guide RNAs to achieve efficient discrimination of a single-nucleotide mutation in rhodopsin ()-P23H mice, a model of dominant retinitis pigmentosa. We found that approximately 45% of the mutant P23H allele was edited at the DNA level and that the relative RNA expression of wild-type was about 2.8 times more than that of mutant in treated retinas. Furthermore, the progression of photoreceptor cell degeneration in outer nuclear layer was significantly delayed in treated regions of the -P23H retinas at 5 weeks of age. Our proof-of-concept study therefore outlines a general strategy that could potentially be expanded to examine the therapeutic benefit of allele-specific gene editing approach to treat human P23H patients. Our study also extends allele-specific editing strategies beyond discrimination within the protospacer adjacent motif sites, with potentially broad applicability to other dominant diseases.
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http://dx.doi.org/10.1089/crispr.2017.0009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319323PMC
February 2018

Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors.

Nat Methods 2017 Dec 30;14(12):1163-1166. Epub 2017 Oct 30.

Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 nuclease fused to transcriptional activation domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.
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http://dx.doi.org/10.1038/nmeth.4483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909187PMC
December 2017

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.

Nature 2017 10 20;550(7676):407-410. Epub 2017 Sep 20.

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

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.
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http://dx.doi.org/10.1038/nature24268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5918688PMC
October 2017

Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.

Oncotarget 2017 Jul;8(27):43733-43751

Department of Medicine, Division of Hematology Oncology, Boston University School of Medicine, Boston, MA, USA.

Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.
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http://dx.doi.org/10.18632/oncotarget.16376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546437PMC
July 2017

Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells.

Nat Biotechnol 2016 08 27;34(8):869-74. Epub 2016 Jun 27.

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

The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9). We also report that four to six bases at the 3' end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.
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http://dx.doi.org/10.1038/nbt.3620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980201PMC
August 2016

Isocitrate Dehydrogenase Mutations Confer Dasatinib Hypersensitivity and SRC Dependence in Intrahepatic Cholangiocarcinoma.

Cancer Discov 2016 07 26;6(7):727-39. Epub 2016 May 26.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts.

Unlabelled: Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy exhibiting frequent isocitrate dehydrogenase (IDH1/IDH2) mutations. Through a high-throughput drug screen of a large panel of cancer cell lines, including 17 biliary tract cancers, we found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multikinase inhibitor dasatinib, with the highest sensitivity among 682 solid tumor cell lines. Using unbiased proteomics to capture the activated kinome and CRISPR/Cas9-based genome editing to introduce dasatinib-resistant "gatekeeper" mutant kinases, we identified SRC as a critical dasatinib target in IDHm ICC. Importantly, dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression. Our results show that IDHm ICC cells have a unique dependency on SRC and suggest that dasatinib may have therapeutic benefit against IDHm ICC. Moreover, these proteomic and genome-editing strategies provide a systematic and broadly applicable approach to define targets of kinase inhibitors underlying drug responsiveness.

Significance: IDH mutations define a distinct subtype of ICC, a malignancy that is largely refractory to current therapies. Our work demonstrates that IDHm ICC cells are hypersensitive to dasatinib and critically dependent on SRC activity for survival and proliferation, pointing to new therapeutic strategies against these cancers. Cancer Discov; 6(7); 727-39. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 681.
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http://dx.doi.org/10.1158/2159-8290.CD-15-1442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458737PMC
July 2016

High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.

Nature 2016 Jan 6;529(7587):490-5. Epub 2016 Jan 6.

Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.
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http://dx.doi.org/10.1038/nature16526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851738PMC
January 2016

Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition.

Nat Biotechnol 2015 Dec 2;33(12):1293-1298. Epub 2015 Nov 2.

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

CRISPR-Cas9 nucleases target specific DNA sequences using a guide RNA but also require recognition of a protospacer adjacent motif (PAM) by the Cas9 protein. Although longer PAMs can potentially improve the specificity of genome editing, they limit the range of sequences that Cas9 orthologs can target. One potential strategy to relieve this restriction is to relax the PAM recognition specificity of Cas9. Here we used molecular evolution to modify the NNGRRT PAM of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, showed robust genome editing activities at endogenous human target sites with NNNRRT PAMs, thereby increasing SaCas9 targeting range by two- to fourfold. Using GUIDE-seq, we show that wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. Our strategy for evolving PAM specificity does not require structural information and therefore should be applicable to a wide range of Cas9 orthologs.
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http://dx.doi.org/10.1038/nbt.3404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689141PMC
December 2015

Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

Nature 2015 Jul 22;523(7561):481-5. Epub 2015 Jun 22.

1] Molecular Pathology Unit &Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [3] Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.
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http://dx.doi.org/10.1038/nature14592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540238PMC
July 2015