CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, Aedes aegypti, a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, kynurenine 3-monooxygenase (kmo), and

In the view of many, heritable human genome editing (HHGE) harbors the remedial potential of ridding the world of deadly genetic diseases. A Hippocratic obligation, if there ever was one, HHGE is widely viewed as a life-sustaining proposition. The national go/no-go decision regarding the implementation of HHGE, however, must not, in the collective view of the authors, proceed absent thorough public

The ability to alter genomes specifically by CRISPR-Cas gene editing has revolutionized biological research, biotechnology, and medicine. Broad therapeutic application of this technology, however, will require thorough preclinical assessment of off-target editing by homology-based prediction coupled with reliable methods for detecting off-target editing. Several off-target site nomination assays exist

Cas12a enzymes are quickly being adopted for use in a variety of genome-editing applications. These programmable nucleases are part of adaptive microbial immune systems, the natural diversity of which has been largely unexplored. Here, we identified novel families of Type V-A CRISPR nucleases through a large-scale analysis of metagenomes collected from a variety of complex environments, and developed

Automated classification of CRISPR-Cas systems has been challenged by their dynamic nature and expanding classification. Here, we developed CRISPRCasTyper, an automated tool with improved capabilities for identifying and typing CRISPR arrays and cas loci based on the latest nomenclature (44 subtypes/variants). As a novel feature, CRISPRCasTyper uses a machine learning approach to subtype CRISPR arrays

We describe a protocol for the precise integration of exogenous DNA into user-defined genomic loci in cultured cells. This strategy first introduces a promoter and a lox site to a specific location via a Cas9-induced double-strand break. Second, a gene of interest (GOI) is inserted into the lox site via Cre-lox recombination. Upon correct insertion, a cis-linked antibiotic resistance gene will be expressed

Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present coronavirus disease 2019 pandemic. Simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, current nucleic acid detection techniques are still limited by traditional amplification and hybridization. To overcome this limitation, here

Among current reported Cas12a orthologs, Francisella novicida Cas12a (FnCas12a) is less restricted by protospacer adjacent motif (PAM). However, the activity of FnCas12a nuclease is relatively low or undetectable in human cells, limiting its application as desirable genome engineering tools. Here, we describe TEXT (Tethering EXonuclease T5 with FnCas12a)—a fusion strategy that significantly increased

Allele-specific genomic targeting by CRISPR is a versatile strategy that has been increasingly exploited not only in treating inherited dominant diseases and mutation-driven cancers, but also in other important fields such as genome imprinting, haploinsufficiency, and genome loci imaging. Despite its tremendous utilities, few bioinformatic tools have been implemented for the allele-specific purpose

Selective breeding and genetic modification have been the cornerstone of animal agriculture. However, the current strategy of breeding animals over multiple generations to introgress novel alleles is not practical in addressing global challenges such as climate change, pandemics, and the predicted need to feed a population of 9 billion by 2050. Consequently, genome editing in zygotes to allow for seamless

CRISPR-Cas systems typically consist of a CRISPR array and cas genes that are organized in one or more operons. However, a substantial fraction of CRISPR arrays are not adjacent to cas genes. Definitive identification of such isolated CRISPR arrays runs into the problem of false-positives, with unrelated types of repetitive sequences mimicking CRISPR. We developed a computational pipeline to eliminate

CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA. Cas9 is revolutionary for the ability to change the RNA sequence and target a new site easily. However, while algorithms

Overweight females are prone to obesity-associated stress urinary incontinence (OA-SUI), and there are no definitive medical therapies for this common urologic condition. This study was designed to test the hypothesis that regenerative therapy to restore urethral striated muscle (stM) and pelvic floor muscles might represent a valuable therapeutic approach. For the in vitro experiment, single-guide

In September 2020, a detailed report on Heritable Human Genome Editing was published. The report offers a translational pathway for the limited approval of germline editing under limited circumstances and assuming various criteria have been met. In this perspective, some three dozen experts from the fields of genome editing, medicine, bioethics, law, and related fields offer their candid reactions

CRISPR-Cas-based transcriptional activators allow genetic engineers to specifically induce expression of one or many target genes in trans. Here we review the many design variations of these versatile tools and compare their effectiveness in different eukaryotic systems. Lastly, we highlight several applications of programmable transcriptional activation to interrogate and engineer complex biological

Discussions and debates about the governance of human germline and heritable genome editing should be informed by a clear and accurate understanding of the global policy landscape. This policy survey of 106 countries yields significant new data. A large majority of countries (96 out of 106) surveyed have policy documents—legislation, regulations, guidelines, codes, and international treaties—relevant

CRISPR-associated proteins 1 and 2 (Cas1–2) are necessary and sufficient for new spacer acquisition in some CRISPR-Cas systems (e.g., type I-E), but adaptation in other systems (e.g., type II-A) involves the crRNA-guided surveillance complex. Here we show that the type I-F Cas1–2/3 proteins are necessary and sufficient to produce low levels of spacer acquisition, but the presence of the type I-F crRNA-guided

CRISPR-based synthetic gene drives have the potential to deliver a more effective and humane method of invasive vertebrate pest control than current strategies. Relatively efficient CRISPR gene drive systems have been developed in insects and yeast but not in mammals. Here, we investigated the efficiency of CRISPR-Cas9-based gene drives in Mus musculus by constructing “split drive” systems where gRNA

CRISPR guide RNAs (gRNAs) can be programmed with relative ease to allow the genetic editing of nearly any DNA or RNA sequence. Here, we propose novel molecular architectures to achieve RNA-dependent modulation of CRISPR activity in response to specific RNA molecules. We designed and tested, in both living Escherichia coli cells and cell-free assays for rapid prototyping, cis-repressed RNA-interacting

The use of CRISPR-Cas and RNA-guided endonucleases has drastically changed research strategies for understanding and exploiting gene function, particularly for the generation of gene-edited animal models. This has resulted in an explosion in the number of gene-edited species, including highly biomedically relevant pig models. However, even with error-free DNA insertion or deletion, edited genes are

This research aims to explore the different ways in which scientists, ethicists, journalists, and commissions speak to the public about new gene-editing technologies. The research collected more than 100,000 sentences from books, news articles, and reports written by these four types of authors, examined the relative distinctiveness of their speech, and compared the prevalence of key terms among the

Over the past 8 years, the widespread adoption of CRISPR-based technologies has fueled the global genome editing revolution. This platform is based on Cas molecular machines such as Cas9, Cas12, Cas13, as well as other CRISPR effector proteins that are able to alter the genome, transcriptome, and epigenome of virtually any species. Technological improvements have rendered these tools more efficient

CRISPR-Cas clinical trials have begun, offering a first glimpse at how DNA and RNA targeting could enable therapies for many genetic and epigenetic human diseases. The speedy progress of CRISPR-Cas from discovery and adoption to clinical use is built on decades of traditional gene therapy research and belies the multiple challenges that could derail the successful translation of these new modalities

Adeno-associated virus (AAV) vectors have been widely adopted for delivery of CRISPR-Cas components, especially for therapeutic gene editing. For a single vector system, both the Cas9 and guide RNA (gRNA) are encoded within a single transgene, usually from separate promoters. Careful design of this bi-cistronic construct is required due to the minimal packaging capacity of AAV. We investigated how

Humans carrying homozygous loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor–related protein 5 (LRP5), develop osteoporosis and a defective retinal vasculature known as familial exudative vitreoretinopathy (FEVR) due to disruption of the Wnt signaling pathway. The purpose of this study was to use CRISPR-Cas9–mediated gene editing to create strains of Lrp5-deficient

RNA interference is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a proof of principle demonstration that a type III Csm effector complex can be used for programmable mRNA transcript degradation in eukaryotes. In zebrafish, Streptococcus thermophilus Csm complex (StCsm) proved effective for knockdown of maternally expressed EGFP in germ cells of

As research on human applications of CRISPR advances, researchers, advisory bodies, and other stakeholder organizations continue calling for global public discourses and engagement to shape the development of human gene editing (HGE). Research that captures public views and tests ways for engaging across viewpoints is vital for facilitating these discourses. Unfortunately, such research lags behind

The principal function of archaeal and bacterial CRISPR-Cas systems is antivirus adaptive immunity. However, recent genome analyses identified a variety of derived CRISPR-Cas variants at least some of which appear to perform different functions. Here, we describe a unique repertoire of CRISPR-Cas-related systems that we discovered by searching archaeal metagenome-assemble genomes of the Asgard superphylum

CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof of principle for Cas ribonuclease RNA targeting in eukaryotic systems

Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency

The CRISPR-Cas9 system can be modified to perform “epigenetic editing” by utilizing the catalytically inactive (dead) Cas9 (dCas9) to recruit regulatory proteins to specific genomic locations. In prior studies, epigenetic editing with multimers of the transactivator VP16 and guide RNAs (gRNAs) was found to cause adverse cellular responses. These side effects may confound studies inducing new cellular

Glycogen synthase kinase 3β (GSK3β) activity is regulated by dopamine D2 receptor signaling and can be inhibited by psychoactive drugs in a D2 receptor–dependent manner. However, GSK3β is ubiquitously expressed in the brain, and D2 receptor–expressing cells are distributed as a mosaic in multiple cortical regions. This complicates the interrogation of GSK3β functions in cortical D2 cells in a circuit-defined

Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. However, some experimental paradigms prove especially challenging and require carefully and appropriately adapted screening approaches. In particular, negative selection (or sensitivity) screening, often the most

CRISPR-Cas has proven to be the most versatile genetic tinkering system of our time, predominantly as a precision genome editing tool. Here, we demonstrate two additions to the repertoire of CRISPR's application for constructing donor DNA templates: CRISPR-CLONInG and CRISPR-CLIP. CRISPR-CLONInG (CRISPR-Cutting and Ligation Of Nucleic acid In vitro via Gibson) was devised to enable efficient cut-and-paste

MAD7 is an engineered class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) system isolated from Eubacterium rectale. Analogous to Cas9, it is an RNA-guided nuclease with demonstrated gene editing activity in Escherichia coli and yeast cells. Here, we report that MAD7 is capable of generating indels and fluorescent gene tagging of endogenous genes in human HCT116 and U2OS cancer cell lines, respectively. In addition

CRISPR/Cas9–based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids

Bemisia tabaci cryptic species Middle East-Asia Minor I (MEAM1) is a serious agricultural polyphagous insect pest and vector of numerous plant viruses, causing major worldwide economic losses. B. tabaci control is limited by lack of robust gene editing tools. Gene editing is difficult in B. tabaci due to small embryos that are technically challenging to inject and which have high mortality post injection

I want to enrich the debate about the ethics and governance of human germline editing (HGE) by emphasizing an underappreciated, yet important, set of concerns regarding exclusionary practices, norms, and efforts that impede a broader discussion about the subject. The possibility for establishing a binding, global, regulatory framework is influenced by economic and geopolitical factors as well as historical

Much of the international community opposes editing the human germline. Yet, given enough experience to become better acquainted with strengths and limitations, prominent international figures are cautiously optimistic about using CRISPR-like novel technologies for clinical applications. Not only might such applications be morally (ethically) permissible, but clinical trials for therapeutic aims could

The case of twins born with genes modified by He Jiankui highlights the need for international governance of germline gene editing (GGE). This article proposes a global framework that utilizes "ordo-responsibilities." This is a pragmatic ethical approach open to pluralism and grounded in principles of human dignity and human rights. Ordo-responsibility is pragmatic in (1) accepting generally available

Plant-breeding technologies have expanded, accelerating breeding research beyond the confines of current regulations. The application of genome editing, such as CRISPR-Cas9, do not neatly fit into existing regulatory frameworks, creating uncertainty as to whether they can be regarded as conventionally developed varieties without further regulation. This research presents the current views of Canadian