Person: Canver, Matthew C.
Loading...
Email Address
AA Acceptance Date
Birth Date
Research Projects
Organizational Units
Job Title
Last Name
Canver
First Name
Matthew C.
Name
Canver, Matthew C.
6 results
Search Results
Now showing 1 - 6 of 6
Publication Human genetic variation alters CRISPR-Cas9 on- and off-targeting specificity at therapeutically implicated loci(National Academy of Sciences, 2017) Lessard, Samuel; Francioli, Laurent; Alfoldi, Jessica; Tardif, Jean-Claude; Ellinor, Patrick; MacArthur, Daniel; Lettre, Guillaume; Orkin, Stuart; Canver, Matthew C.The CRISPR-Cas9 nuclease system holds enormous potential for therapeutic genome editing of a wide spectrum of diseases. Large efforts have been made to further understanding of on- and off-target activity to assist the design of CRISPR-based therapies with optimized efficacy and safety. However, current efforts have largely focused on the reference genome or the genome of cell lines to evaluate guide RNA (gRNA) efficiency, safety, and toxicity. Here, we examine the effect of human genetic variation on both on- and off-target specificity. Specifically, we utilize 7,444 whole-genome sequences to examine the effect of variants on the targeting specificity of ∼3,000 gRNAs across 30 therapeutically implicated loci. We demonstrate that human genetic variation can alter the off-target landscape genome-wide including creating and destroying protospacer adjacent motifs (PAMs). Furthermore, single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) can result in altered on-target sites and novel potent off-target sites, which can predispose patients to treatment failure and adverse effects, respectively; however, these events are rare. Taken together, these data highlight the importance of considering individual genomes for therapeutic genome-editing applications for the design and evaluation of CRISPR-based therapies to minimize risk of treatment failure and/or adverse outcomes.Publication Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci(2017) Canver, Matthew C.; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily; Needleman, Austen; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin; Reid, Marvin; Chen, Diane D.; Das, Partha Pratim; Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel; Orkin, StuartCas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants from genome-wide association studies largely cluster in regulatory DNA. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating mutagenesis libraries with single or combinatorial nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, a locus associated with red blood cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false positive regions, which emphasizes the importance of off-target analysis in design of saturating mutagenesis experiments. Taken together, these data establish a widely applicable high-throughput and high-resolution methodology to reliably identify minimal functional sequences within large regions of disease- and trait-associated DNA.Publication BCL11A Enhancer Dissection by Cas9-Mediated in Situ Saturating Mutagenesis(Nature, 2015-09-16) Canver, Matthew C.; Smith, Elenoe; Pinello, Luca; Sanjana, Neville E.; Shalem, Ophir; Chen, Diane; Schupp, Patrick G.; Vinjamur, Divya S.; Garcia, Sara P.; Luc, Sidinh; Kurita, Ryo; Fujiwara, Yuko; Maeda, Takahiro; Yuan, Guo-Cheng; Zhang, Feng; Orkin, Stuart; Bauer, Daniel; Nakamura, YukioPublication Angiopoietin-like proteins stimulate HSPC development through interaction with notch receptor signaling(eLife Sciences Publications, Ltd, 2015) Lin, Michelle I; Price, Emily N; Boatman, Sonja; Hagedorn, Elliott; Trompouki, Eirini; Satishchandran, Sruthi; Carspecken, Charles W; Uong, Audrey; DiBiase, Anthony; Yang, Song; Canver, Matthew C.; Dahlberg, Ann; Lu, Zhigang; Zhang, Cheng Cheng; Orkin, Stuart; Bernstein, Irwin D; Aster, Jon C; White, Richard M; Zon, LeonardAngiopoietin-like proteins (angptls) are capable of ex vivo expansion of mouse and human hematopoietic stem and progenitor cells (HSPCs). Despite this intriguing ability, their mechanism is unknown. In this study, we show that angptl2 overexpression is sufficient to expand definitive HSPCs in zebrafish embryos. Angptl1/2 are required for definitive hematopoiesis and vascular specification of the hemogenic endothelium. The loss-of-function phenotype is reminiscent of the notch mutant mindbomb (mib), and a strong genetic interaction occurs between angptls and notch. Overexpressing angptl2 rescues mib while overexpressing notch rescues angptl1/2 morphants. Gene expression studies in ANGPTL2-stimulated CD34+ cells showed a strong MYC activation signature and myc overexpression in angptl1/2 morphants or mib restored HSPCs formation. ANGPTL2 can increase NOTCH activation in cultured cells and ANGPTL receptor interacted with NOTCH to regulate NOTCH cleavage. Together our data provide insight to the angptl-mediated notch activation through receptor interaction and subsequent activation of myc targets. DOI: http://dx.doi.org/10.7554/eLife.05544.001Publication Elucidation of Mechanisms of Fetal Hemoglobin Regulation by CRISPR/Cas9 Mediated Genome Editing(2016-03-14) Canver, Matthew C.; Joung, Jae; Kuroda, Mitzi; Sontheimer, ErikDespite nearly complete understanding of the genetics of the β-hemoglobinopathies for several decades, definitive treatment options have lagged behind. Fetal hemoglobin (HbF) reinduction represents a “silver bullet” for therapy of the β-globin disorders. Recent development of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease system has allowed for facile manipulation of the genome for the study of genes and genetic elements. Here we developed CRISPR/Cas9-based methodology to reliably engender targeted genomic deletions ranging from 1.3 kilobases to over 1 megabase, which suggested an inverse relationship between deletion size and deletion frequency. Targeted deletion methods and Cas9-mediated in situ saturating mutagenesis were applied to the enhancer of the HbF repressor BCL11A, which revealed discrete vulnerabilities. This finding is consistent with emerging evidence in the field that large enhancers are comprised of constituent parts with some harboring the majority of the activity. The identified “Achilles heel” of the enhancer represents a promising therapeutic target. We further enhanced the resolution of the in situ saturating mutagenesis technique by using multiple Cas9 nucleases and variant-aware library design to identify functional sequences within the HBS1L-MYB intergenic region, a locus associated with elevated HbF levels. These data demonstrate the robustness of CRISPR/Cas9 mediated in situ saturating mutagenesis and targeted deletion to interrogate functional sequence within regulatory DNA. Harnessing the power of genome editing may usher in a second generation form of gene therapy for the β-globin disorders.Publication The Effect of Human Genetic Variation on CRISPR Targeting Specificity(2018-05-15) Canver, Matthew C.The clustered regularly interspaced short palindromic repeats (CRISPR) nuclease system offers the ability for unprecedented functional genetic experiments and the promise for therapy of a variety of genetic disorders. The understanding of factors contributing to CRISPR targeting efficacy and specificity continue to evolve. As CRISPR systems rely on Watson-Crick base pairing to ultimately mediate genomic cleavage, it logically follows that genetic variation would affect CRISPR targeting by increasing or decreasing sequence homology at on- and off-target sites or by altering protospacer adjacent motifs (PAMs). Numerous efforts have been made to document the extent of human genetic variation, which serve as resources to understand and mitigate the effect of genetic variation on CRISPR targeting. Here, we examine the effect of human genetic variation on CRISPR targeting at on-target and off-target sites with consideration for clinical translation of CRISPR-based therapies.