Dennis Grishin

Bio: Dennis Grishin is an academic researcher from Harvard University. The author has contributed to research in topics: Genome-wide association study & Information privacy. The author has an hindex of 6, co-authored 8 publications receiving 769 citations.

Genome-wide inactivation of porcine endogenous retroviruses (PERVs)

Abstract: The shortage of organs for transplantation is a major barrier to the treatment of organ failure. Although porcine organs are considered promising, their use has been checked by concerns about the transmission of porcine endogenous retroviruses (PERVs) to humans. Here we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all copies of the PERV pol gene and demonstrated a >1000-fold reduction in PERV transmission to human cells, using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine-to-human xenotransplantation.

‘Fit-for-purpose?’ – challenges and opportunities for applications of blockchain technology in the future of healthcare

Abstract: Blockchain is a shared distributed digital ledger technology that can better facilitate data management, provenance and security, and has the potential to transform healthcare. Importantly, blockchain represents a data architecture, whose application goes far beyond Bitcoin – the cryptocurrency that relies on blockchain and has popularized the technology. In the health sector, blockchain is being aggressively explored by various stakeholders to optimize business processes, lower costs, improve patient outcomes, enhance compliance, and enable better use of healthcare-related data. However, critical in assessing whether blockchain can fulfill the hype of a technology characterized as ‘revolutionary’ and ‘disruptive’, is the need to ensure that blockchain design elements consider actual healthcare needs from the diverse perspectives of consumers, patients, providers, and regulators. In addition, answering the real needs of healthcare stakeholders, blockchain approaches must also be responsive to the unique challenges faced in healthcare compared to other sectors of the economy. In this sense, ensuring that a health blockchain is ‘fit-for-purpose’ is pivotal. This concept forms the basis for this article, where we share views from a multidisciplinary group of practitioners at the forefront of blockchain conceptualization, development, and deployment.

Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells

Abstract: CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in humaninduced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Wholegenome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be B1.5–8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design.

Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.

Abstract: Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.

Accelerating Genomic Data Generation and Facilitating Genomic Data Access Using Decentralization, Privacy-Preserving Technologies and Equitable Compensation

Abstract: In the years since the first human genome was sequenced at a cost of over $3 billion, technological advancements have driven the price below $1,000, making personal genome sequencing affordable to many people. Personal genome sequencing has the potential to enable better disease prevention, more accurate diagnoses, and personalized therapies. Furthermore, sharing genomic data with researchers promises identification of the causes of many diseases and the development of new therapies. However, sequencing costs, data privacy concerns, regulatory restrictions, and technical challenges impede the growth of genomic data and hinder data sharing. In this article, we propose that these challenges can be addressed by combining decentralized system design, privacy-preserving technologies, and an equitable compensation model in a platform that vests control over data with individual owners; ensures transparency and privacy; facilitates regulatory compliance; minimizes expensive data transfers; and shifts the sequencing costs from consumers, patients, and biobanks to researchers in industry and academia. We exemplify this by describing the implementation of Nebula, a distributed genomic data generation, sharing, and analysis platform.

Integrative Genomics Viewer

Abstract: Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

The knowledge complexity of interactive proof-systems

Abstract: Usually, a proof of a theorem contains more knowledge than the mere fact that the theorem is true. For instance, to prove that a graph is Hamiltonian it suffices to exhibit a Hamiltonian tour in it; however, this seems to contain more knowledge than the single bit Hamiltonian/non-Hamiltonian.In this paper a computational complexity theory of the “knowledge” contained in a proof is developed. Zero-knowledge proofs are defined as those proofs that convey no additional knowledge other than the correctness of the proposition in question. Examples of zero-knowledge proof systems are given for the languages of quadratic residuosity and 'quadratic nonresiduosity. These are the first examples of zero-knowledge proofs for languages not known to be efficiently recognizable.

Therapeutic genome editing: prospects and challenges

Abstract: Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.

Therapeutic genome editing: prospects and challenges

Abstract: Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.