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Methods for In Vivo CRISPR/Cas Editing of the Adult Murine Retina.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) is used by some bacteria and most archaea to protect against viral phage intrusion and has recently been adapted to allow for efficient editing of the mammalian genome. Whilst CRISPR/Cas-based technology has been used to modify genes in mammalian cells in vitro, delivery of CRISPR/Cas system into mammalian tissue and/or organs is more difficult and often requires additional vectors. With the use of adeno-associated virus (AAV) gene delivery system, active CRISPR/Cas enzyme can be maintained for an extended period of time and enable efficient editing of genome in the retina in vivo. Herein we outline the method to edit the genome in mouse retina using a dual AAV vector -mediated CRISPR/Cas9 system.
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CRISPR/Cas9 for Human Genome Engineering and Disease Research.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, a versatile RNA-guided DNA targeting platform, has been revolutionizing our ability to modify, manipulate, and visualize the human genome, which greatly advances both biological research and therapeutics development. Here, we review the current development of CRISPR/Cas9 technologies for gene editing, transcription regulation, genome imaging, and epigenetic modification. We discuss the broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases.
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Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine.
INTRODUCTION: Clustered regularly interspaced short palindromic repeats (CRISPR) are segments of nucleic acid that play a role in prokaryotic defense and form the basis of a genome editing technology that allows permanent alteration of genetic material. This methodology, known as CRISPR-Cas9, is poised to revolutionize molecular biology, but no literature yet exists on how these advances will affect hospitalists. AREAS COVERED: These specialists in inpatient medicine care for a wide variety of hospitalized patients, including those with infectious disease, cancer, cardiovascular disease, autoimmune disease, hematologic disease, and a variety of other conditions that may soon be impacted by advances in gene-modifying technology provided by CRISPR-Cas9. A Literature search was performed using PubMed [1 December 2019-17 April 2020]. EXPERT OPINION: This paper reviews the remarkable diagnostic and therapeutic potential of the CRISPR-Cas9 platform and concludes with a look at ethical issues and technical hurdles pertaining to the implementation of permanent gene modification in the practice of Hospital Medicine.
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Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines.
The CRISPR/Cas has been recently shown to be a powerful genome-editing tool in a variety of organisms. However, these studies are mainly focused on protein-coding genes. The present study aims to determine whether this technology can be applied to non-coding genes. One of the challenges for knockout of non-coding genes is that a small deletion or insertion generated by the standard CRISPR/Cas system may not necessarily lead to functional loss of a given non-coding gene because of lacking an open reading frame, especially in polyploidy human cell lines. To overcome this challenge, we adopt a selection system that allows for marker genes to integrate into the genome through homologous recombination (HR). Moreover, we construct a dual guide RNA vector that can make two cuts simultaneously at designated sites such that a large fragment can be deleted. With these approaches, we are able to successfully generate knockouts for miR-21, miR-29a, lncRNA-21A, UCA1 and AK023948 in various human cell lines. Finally, we show that the HR-mediated targeting efficiency can be further improved by suppression of the non-homologous end joining pathway. Together, these results demonstrate the feasibility of knockout for non-coding genes by the CRISPR/Cas system in human cell lines.
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Enhanced Golic+: highly effective CRISPR gene targeting and transgene HACKing in Drosophila.
Gene targeting is an incredibly valuable technique. Sometimes, however, it can also be extremely challenging for various intrinsic reasons (e.g. low target accessibility or nature/extent of gene modification). To bypass these barriers, we designed a transgene-based system in Drosophila that increases the number of independent gene targeting events while at the same time enriching for correctly targeted progeny. Unfortunately, with particularly challenging gene targeting experiments, our original design yielded numerous false positives. Here, we deliver a much-improved technique, named Enhanced Golic+ (E-Golic+). E-Golic+ incorporates genetic modifications to tighten lethality-based selection while simultaneously boosting efficiency. With E-Golic+, we easily achieve previously unattainable gene targeting. Additionally, we built an E-Golic+-based, high-efficiency genetic pipeline for transgene swapping. We demonstrate its utility by transforming GAL4 enhancer-trap lines into tissue-specific Cas9-expressing lines. Given the superior efficiency, specificity and scalability, E-Golic+ promises to expedite development of additional sophisticated genetic/genomic tools in Drosophila.
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Strategies for In Vivo Genome Editing in Nondividing Cells.
Programmable nucleases, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), have enhanced our ability to edit genomes by the sequence-specific generation of double-strand breaks (DSBs) with subsequent homology-directed repair (HDR) of the DSB. However, the efficiency of the HDR pathway is limited in nondividing cells, which encompass most of the cells in the body. Therefore, the HDR-mediated genome-editing approach has limited in vivo applicability. Here, we discuss a mutation type-oriented viewpoint of strategies devised over the past few years to circumvent this problem, along with their possible applications and limitations.
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Therapeutic genome engineering via CRISPR-Cas systems.
Differences in genomes underlie most organismal diversity, and aberrations in genomes underlie many disease states. With the growing knowledge of the genetic and pathogenic basis of human disease, development of safe and efficient platforms for genome and epigenome engineering will transform our ability to therapeutically target human diseases and also potentially engineer disease resistance. In this regard, the recent advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) RNA-guided nuclease systems have transformed our ability to target nucleic acids. Here we review therapeutic genome engineering applications with a specific focus on the CRISPR-Cas toolsets. We summarize past and current work, and also outline key challenges and future directions. WIREs Syst Biol Med 2017, 9:e1380. doi: 10.1002/wsbm.1380 For further resources related to this article, please visit the WIREs website.
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Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity.
Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2',4'-BNANC[N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNANC incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA-DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, "zipped" conformation. In addition to describing a robust technique for improving the precision of CRISPR/Cas9-based gene editing, this study illuminates an application of synthetic nucleic acids.
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CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos.
Conventional embryonic stem cell (ESC)-based gene targeting, zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.
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Expanding the genetic toolkit in Xenopus: Approaches and opportunities for human disease modeling.
The amphibian model Xenopus, has been used extensively over the past century to study multiple aspects of cell and developmental biology. Xenopus offers advantages of a non-mammalian system, including high fecundity, external development, and simple housing requirements, with additional advantages of large embryos, highly conserved developmental processes, and close evolutionary relationship to higher vertebrates. There are two main species of Xenopus used in biomedical research, Xenopus laevis and Xenopus tropicalis; the common perception is that both species are excellent models for embryological and cell biological studies, but only Xenopus tropicalis is useful as a genetic model. The recent completion of the Xenopus laevis genome sequence combined with implementation of genome editing tools, such as TALENs (transcription activator-like effector nucleases) and CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated nucleases), greatly facilitates the use of both Xenopus laevis and Xenopus tropicalis for understanding gene function in development and disease. In this paper, we review recent advances made in Xenopus laevis and Xenopus tropicalis with TALENs and CRISPR-Cas and discuss the various approaches that have been used to generate knockout and knock-in animals in both species. These advances show that both Xenopus species are useful for genetic approaches and in particular counters the notion that Xenopus laevis is not amenable to genetic manipulations.
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