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CRATES: A one-step assembly method for Class 2 CRISPR arrays.
CRISPR-Cas systems naturally rely on CRISPR arrays to achieve immunity against multiple foreign invaders, where these arrays are also being utilized for multiplexed targeting as part of CRISPR technologies. However, CRISPR arrays have proven difficult to synthesize or assemble to-date due to the repetitive DNA repeats in each array. To overcome this barrier, we recently reported a cloning method we term CRATES (CRISPR Assembly through Trimmed Ends of Spacers) for the single-step, efficient generation of large Class 2 CRISPR arrays. CRATES generates CRISPR arrays through assembly of multiple repeat-spacer subunits using defined junction sequences within the trimmed portion of the CRISPR spacers. These arrays can be utilized by single-effector nucleases associated with Class 2 CRISPR-Cas systems, such as Cas9, Cas12a/Cpf1, or Cas13a/C2c2. Here, we describe in detail the steps for generating arrays utilized by Cas9 and Cas12a as well as composite arrays co-utilized by both nucleases. We also generate a representative three-spacer array and demonstrate multiplexed DNA cleavage through plasmid-clearance assays in Escherichia coli. This method is expected to simplify the study of natural CRISPR arrays and facilitate multiplexed targeting with programmable nucleases from Class 2 Cas nucleases across the myriad applications of CRISPR technologies.
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CRISPR Genome Editing Made Easy Through the CHOPCHOP Website.
The design of optimal guide RNA (gRNA) sequences for CRISPR systems is challenged by the need to achieve highly efficient editing at the desired location (on-target editing) with minimal editing at unintended locations (off-target editing). Although laboratory validation should ideally be used to detect off-target activity, computational predictions are almost always preferred in practice due to their speed and low cost. Several studies have therefore explored gRNA-DNA interactions in order to understand how CRISPR complexes select their genomic targets. CHOPCHOP (https://chopchop.cbu.uib.no/) leverages these developments to build a user-friendly web interface that helps users design optimal gRNAs. CHOPCHOP supports a wide range of CRISPR applications, including gene knock-out, sequence knock-in, and RNA knock-down. Furthermore, CHOPCHOP offers visualization that enables an informed choice of gRNAs and supports experimental validation. In these protocols, we describe the best practices for gRNA design using CHOPCHOP. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of gRNAs for gene knock-out Alternate Protocol 1: Design of gRNAs for dCas9 fusion/effector targeting Support Protocol: Design of gRNAs for targeting transgenic or plasmid sequences Basic Protocol 2: Design of gRNAs for RNA targeting Basic Protocol 3: Design of gRNAs for sequence knock-in Alternate Protocol 2: Design of gRNAs for knock-in using non-homologous end joining Basic Protocol 4: Design of gRNAs for knock-in using Cas9 nickases.
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What we know about effective public engagement on CRISPR and beyond.
Advances in gene editing technologies for human, plant, and animal applications have led to calls from bench and social scientists, as well as a wide variety of societal stakeholders, for broad public engagement in the decision-making about these new technologies. Unfortunately, there is limited understanding among the groups calling for public engagement on CRISPR and other emerging technologies about 1) the goals of this engagement, 2) the modes of engagement and what we know from systematic social scientific evaluations about their effectiveness, and 3) how to connect the products of these engagement exercises to societal decision or policy making. Addressing all three areas, we systematize common goals, principles, and modalities of public engagement. We evaluate empirically the likely successes of various modalities. Finally, we outline three pathways forward that deserve close attention from the scientific community as we navigate the world of Life 2.0.
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Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7.
The CRISPR-Cas9 system has become increasingly popular for genome engineering across all fields of biological research, including in the Gram-positive model organism Bacillus subtilis. A major drawback for the commercial use of Cas9 is the IP landscape requiring a license for its use, as well as reach-through royalties on the final product. Recently an alternative CRISPR nuclease, free to use for industrial R&D, MAD7 was released by Inscripta (CO). Here we report the first use of MAD7 for gene editing in B. subtilis, in which editing rates of 93% and 100% were established. Additionally, we engineer the first reported catalytically inactive MAD7 (dMAD7) variant (D877A, E962A, and D1213A) and demonstrate its utility for CRISPR interference (CRISPRi) at up to 71.3% reduction of expression at single and multiplexed target sites within B. subtilis. We also confirm the CRISPR-based editing mode of action in B. subtilis providing evidence that the nuclease-mediated DNA double-strand break acts as a counterselection mechanism after homologous recombination of the donor DNA.
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Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos.
This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm-40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.
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CRISPR-Cas9 Genome Interrogation: A Facilitated Subdiffusive Target Search Strategy.
The functional application of RNA-guided CRISPR-associated Cas9 protein, a bacterial immune system-based protein complex, via which in vivo, highly specific, and well-regulated, gene-editing processes are being monitored at an unprecedented level, has led to remarkable progress in genetic engineering and technology. The complicated in vivo process of genome interrogation followed by gene editing by the Cas9 complex was recently reported by Knight et al. (Science, 2015, 350, 823-826) using an elegant single-particle tracking method, aided by the two-photon fluorescence correlation spectroscopic technique. In contrast to the usually observed fast target-searching and protein-binding events in biophysical systems, an interesting slow genome-interrogation process by the RNA-guided CRISPR-Cas9 system through a crowded chromatin environment of a mammalian cell has been revealed in Knight et al.'s study. Motivated by this experiment, in this paper, we provide a generalized theoretical framework to capture this particular target-searching mechanism of the CRISPR-Cas9 protein complex. We show that an analysis on the basis of 3D subdiffusion under a cylindrical volume, created by several nonspecific off-target interactions from the DNA strands, can capture the essential details of the process. Moreover, on the basis of this model, we quantify the dynamics of this process and estimate the survival probability, first passage time, and the intensity correlation function of the tagged proteins of the experiment. The results from our theoretical predictions are found to be consistent with the experimental observations, and hence, seem to provide a plausible microscopic picture of the process.
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Characterization and Repurposing of Type I and Type II CRISPR-Cas Systems in Bacteria.
CRISPR-Cas systems constitute the adaptive immune system of bacteria and archaea, as a sequence-specific nucleic acid targeting defense mechanism. The sequence-specific recognition and cleavage of Cas effector complexes has been harnessed to developed CRISPR-based technologies and drive the genome editing revolution underway, due to their efficacy, efficiency, and ease of implementation in a broad range of organisms. CRISPR-based technologies offer a wide variety of opportunities in genome remodeling and transcriptional regulation, opening new avenues for therapeutic and biotechnological applications. To repurpose CRISPR-Cas systems for these applications, the various elements of the system need to be first identified and functionally characterized in their native host. Bioinformatic tools are first used to identify putative CRISPR arrays and their associated genes, followed by a comprehensive characterization of the CRISPR-Cas system, encompassing predictions for guide and target sequences. Subsequently, interference assays and transcriptomic analyses should be performed to probe the functionality of the CRISPR-Cas system. Once an endogenous CRISPR-Cas system is characterized as functional, they can be readily repurposed by delivering an engineered synthetic CRISPR array or a small RNA guide for targeted gene manipulation. Alternatively, developing a plasmid-based system for heterologous expression of the necessary CRISPR components can enable exploitation in other organisms. Altogether, there is a wide diversity of native CRISPR-Cas systems in many bacteria and most archaea that await functional characterization and repurposing for genome editing applications in prokaryotes.
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Targeting epigenetic aberrations of sarcoma in CRISPR era.
Sarcomas are rare malignancies that exhibit diverse biological, genetic, morphological, and clinical characteristics. Genetic alterations, such as gene fusions, mutations in transcriptional machinery components, histones, and DNA methylation regulatory molecules, play an essential role in sarcomagenesis. These mutations induce and/or cooperate with specific epigenetic aberrations required for the growth and maintenance of sarcomas. Appropriate mouse models have been developed to clarify the significance of genetic and epigenetic interactions in sarcomas. Studies using the mouse models for human sarcomas have demonstrated major advances in our understanding the developmental processes as well as tumor microenvironment of sarcomas. Recent technological progresses in epigenome editing will not only improve the studies using animal models but also provide a direct clue for epigenetic therapies. In this manuscript, we review important epigenetic aberrations in sarcomas and their representative mouse models, current methods of epigenetic editing using CRISPR/dCas9 systems, and potential applications in sarcoma studies and therapeutics.
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Protocol for non-invasive delivery of CRISPR RNPs via virus-like particles for mouse model generation.
CRISPR-virus-like particle (VLP)-induced targeted mutagenesis (CRISPR-VIM) enables genome editing in mouse embryos through non-invasive delivery of CRISPR ribonucleoproteins (RNPs) via VLPs, eliminating the need for physical manipulation and specialized expertise. We detail protocols for VLP production, titration, and treatment for diverse genome edits. This protocol is compatible with zygotes and in vitro fertilization (IVF)-derived embryos via simple co-culture, facilitating high-efficiency and heritable mutations with minimized off-target effects, independent of specialized equipment and conducive to reduced animal use. For complete details on the use and execution of this protocol, please refer to Jeong et al.1.
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i-CRISPR: a personalized cancer therapy strategy through cutting cancer-specific mutations.
Developing a strategy to specifically kill cancer cells without inducing obvious damage to normal cells may be of great clinical significance for cancer treatment. In the present study, we developed a new precise personalized strategy named "i-CRISPR" for cancer treatment through adding DNA damage repair inhibitors(i) and inducing cancer cell-specific DNA double strand breaks by CRISPR. Through in vitro and in vivo experiments, we confirmed the efficacy of this strategy in multiple cancer models and revealed the mechanism of cell death. Our strategy might provide a novel concept for precise cancer therapy.
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