CrispRVariants: precisely charting the mutation spectrum in genome engineering experiments

Mapping Intimacies ◽

10.1101/034140 ◽

2015 ◽

Cited By ~ 1

Author(s):

Helen Lindsay ◽

Alexa Burger ◽

Berthin Biyong ◽

Anastasia Felker ◽

Christopher Hess ◽

...

Keyword(s):

Genomic Dna ◽

Gene Editing ◽

Genome Engineering ◽

Careful Consideration ◽

Functional Screening ◽

Sequencing Data ◽

Web Tool ◽

Therapeutic Gene ◽

Accurate Evaluation ◽

Mutagenic Process

CRISPR-Cas9 and related technologies efficiently alter genomic DNA at targeted positions and have far-reaching implications for functional screening and therapeutic gene editing. Understanding and unlocking this potential requires accurate evaluation of editing efficiency. We show that methodological decisions for analyzing sequencing data can significantly affect mutagenesis efficiency estimates and we provide a comprehensive R-based toolkit, CrispRVariants and accompanying web tool CrispRVariantsLite, that resolves and localizes individual mutant alleles with respect to the endonuclease cut site. CrispRVariants-enabled analyses of newly generated and existing genome editing datasets underscore how careful consideration of the full variant spectrum gives insight toward effective guide and amplicon design as well as the mutagenic process.

Download Full-text

Automated design of CRISPR prime editors for thousands of human pathogenic variants

10.1101/2020.05.07.083444 ◽

2020 ◽

Author(s):

John A. Morris ◽

Jahan A. Rahman ◽

Xinyi Guo ◽

Neville E. Sanjana

Keyword(s):

Genetic Variants ◽

Gene Editing ◽

Genome Engineering ◽

Disease Modeling ◽

Automated Design ◽

Common Variants ◽

Therapeutic Gene ◽

Web Based ◽

Pathogenic Variants ◽

Automated Pipeline

AbstractPrime editors (PEs) are CRISPR-based genome engineering tools that can introduce precise base-pair edits at specific locations in the genome. These programmable gene editors have been predicted to repair 89% of known human pathogenic variants in the ClinVar database, although these PE constructs do not presently exist. Towards this end, we developed an automated pipeline to correct (therapeutic editing) or introduce (disease modeling) human pathogenic variants that optimizes the design of several RNA constructs required for prime editing and avoids predicted off-targets in the human genome. However, using optimal PE design criteria, we find that only a small fraction of these pathogenic variants can be targeted. Through the use of alternative Cas9 enzymes and extended templates, we increase the number of targetable pathogenic variants to >50,000 variants and make these pre-designed PE constructs accessible through a web-based portal (http://primeedit.nygenome.org). Given the tremendous potential for therapeutic gene editing, we also assessed the possibility of developing universal PE constructs. By examining the overlap of different PE components with common human genetic variants in dbSNP, we find that common variants affect only a small minority of designed PEs.

Download Full-text

Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

International Journal of Genomics ◽

10.1155/2014/434575 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8

Author(s):

Momchilo Vuyisich ◽

Ayesha Arefin ◽

Karen Davenport ◽

Shihai Feng ◽

Cheryl Gleasner ◽

...

Keyword(s):

Genomic Dna ◽

De Novo ◽

Gc Content ◽

Library Preparation ◽

Sequencing Data ◽

Bacterial Genomes ◽

Dna Amount ◽

High Quality ◽

Preparation Methods

Sequencing bacterial genomes has traditionally required large amounts of genomic DNA (~1 μg). There have been few studies to determine the effects of the input DNA amount or library preparation method on the quality of sequencing data. Several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input DNA. In this study, we evaluated the NEBNext Ultra library preparation reagents for sequencing bacterial genomes. We have evaluated the utility of NEBNext Ultra for resequencing andde novoassembly of four bacterial genomes and compared its performance with the TruSeq library preparation kit. The NEBNext Ultra reagents enable high quality resequencing andde novoassembly of a variety of bacterial genomes when using 100 ng of input genomic DNA. For the two most challenging genomes (Burkholderiaspp.), which have the highest GC content and are the longest, we also show that the quality of both resequencing andde novoassembly is not decreased when only 10 ng of input genomic DNA is used.

Download Full-text

Population sequencing data reveal a compendium of mutational processes in the human germ line

Science ◽

10.1126/science.aba7408 ◽

2021 ◽

pp. eaba7408

Author(s):

Vladimir B. Seplyarskiy ◽

Ruslan A. Soldatov ◽

Evan Koch ◽

Ryan J. McGinty ◽

Jakob M. Goldmann ◽

...

Keyword(s):

Germ Line ◽

Nonnegative Matrix ◽

Replication Timing ◽

Mutagenic Effect ◽

Dna Lesions ◽

Sequencing Data ◽

Biological Interpretation ◽

Mutagenic Process ◽

Mutational Processes ◽

Transcription And Replication

Biological mechanisms underlying human germline mutations remain largely unknown. We statistically decompose variation in the rate and spectra of mutations along the genome using volume-regularized nonnegative matrix factorization. The analysis of a sequencing dataset (TOPMed) reveals nine processes that explain the variation in mutation properties between loci. We provide a biological interpretation for seven of these processes. We associate one process with bulky DNA lesions that resolve asymmetrically with respect to transcription and replication. Two processes track direction of replication fork and replication timing, respectively. We identify a mutagenic effect of active demethylation primarily acting in regulatory regions and a mutagenic effect of LINE repeats. We localize a mutagenic process specific to oocytes from population sequencing data. This process appears transcriptionally asymmetric.

Download Full-text

Therapeutic Gene Editing with CRISPR

Clinics in Laboratory Medicine ◽

10.1016/j.cll.2020.02.008 ◽

2020 ◽

Vol 40 (2) ◽

pp. 205-219

Author(s):

Elan Hahn ◽

Matthew Hiemenz

Keyword(s):

Gene Editing ◽

Therapeutic Gene

Download Full-text

CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy

Cancer Cell International ◽

10.1186/s12935-020-01546-8 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 1

Author(s):

Seyed Mohammad Miri ◽

Elham Tafsiri ◽

William Chi Shing Cho ◽

Amir Ghaemi

Keyword(s):

T Cells ◽

Cancer Immunotherapy ◽

Gene Editing ◽

Genome Engineering ◽

Building Blocks ◽

Cell Receptor ◽

Adoptive Cell Transfer ◽

Promising Strategy ◽

Pre Treatment ◽

Cas A

Abstract Cancer immunotherapy has been emerged as a promising strategy for treatment of a broad spectrum of malignancies ranging from hematological to solid tumors. One of the principal approaches of cancer immunotherapy is transfer of natural or engineered tumor-specific T-cells into patients, a so called “adoptive cell transfer”, or ACT, process. Construction of allogeneic T-cells is dependent on the employment of a gene-editing tool to modify donor-extracted T-cells and prepare them to specifically act against tumor cells with enhanced function and durability and least side-effects. In this context, CRISPR technology can be used to produce universal T-cells, equipped with recombinant T cell receptor (TCR) or chimeric antigen receptor (CAR), through multiplex genome engineering using Cas nucleases. The robust potential of CRISPR-Cas in preparing the building blocks of ACT immunotherapy has broaden the application of such therapies and some of them have gotten FDA approvals. Here, we have collected the last investigations in the field of immuno-oncology conducted in partnership with CRISPR technology. In addition, studies that have addressed the challenges in the path of CRISPR-mediated cancer immunotherapy, as well as pre-treatment applications of CRISPR-Cas have been mentioned in detail.

Download Full-text

Call for Papers: Expanding the Scale and Scope of Therapeutic Gene Editing

Molecular Therapy ◽

10.1016/j.ymthe.2020.07.007 ◽

2020 ◽

Vol 28 (8) ◽

pp. 1743

Author(s):

Hans-Peter Kiem ◽

Nicole N. Gaudelli ◽

Fyodor D. Urnov ◽

Robert M. Frederickson ◽

Roland W. Herzog

Keyword(s):

Gene Editing ◽

Therapeutic Gene

Download Full-text

Advances in therapeutic application of CRISPR-Cas9

Briefings in Functional Genomics ◽

10.1093/bfgp/elz031 ◽

2019 ◽

Vol 19 (3) ◽

pp. 164-174 ◽

Cited By ~ 3

Author(s):

Jinyu Sun ◽

Jianchu Wang ◽

Donghui Zheng ◽

Xiaorong Hu

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Malignant Tumor ◽

Gene Editing ◽

Genome Engineering ◽

Therapeutic Application ◽

Adaptive Immune ◽

Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

Download Full-text