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

In the midst of the media and professional exuberance regarding the potential benefits of CRISPR technology, voices of criticism and caution have also arisen. One of the thorniest such cautions has been the common objection that CRISPR allows bioscientists to "play God," particularly when it comes to potentially editing the human germline. Many in the biotechnology field are unsure how to address this

The potential for using germline genome editing (GGE) in humans has garnered a lot of attention, both for its scientific possibilities as well as for the ethical, legal, and social challenges it ignites. The ethical debate has focused primarily on the suggestions of using GGE to establish a pregnancy (i.e., to offer it in a clinical setting), which is, to date, illegal in many jurisdictions. The use

If the safety and efficacy issues relating to heritable genome editing can be resolved, how should liberal democratic societies regulate the use of this technology by prospective parents who wish to effect edits to the genomes of their prospective children? We suggest that recent developments in South African law can be useful in this regard. The country's apex court recently recognized as a legal

The social acceptability of germline genome editing (GGE) depends on its perceived safety, as well as respect for reproductive autonomy. However, it is doubtful that prospective parents sufficiently understand the risks of GGE. In the future, the use of GGE in specific situations seems plausible, as it offers couples potential means to safeguard genetically related future children from a serious disease

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer

Efficient site-directed insertion of heterologous DNA into a genome remains an outstanding challenge. Recombinases that can integrate kilobase-sized DNA constructs are difficult to reprogram to user-defined loci, while genomic insertion using CRISPR-Cas methods relies on inefficient host DNA repair machinery. Here, we describe a Cas-Transposon (CasTn) system for genomic insertions that uses a Himar1

CRISPR-Cas9 has quickly become the method of choice for genome editing, with multiple publications describing technical advances and novel applications. It has been widely adopted as a tool for basic research and has significant translational and clinical potential. However, its usage has outpaced the establishment of essential and rigorous controls for unwanted off-target effects, manifested as small

Advances in CRISPR technology and the announcement of the first gene-edited babies have sparked a global dialogue about the future of heritable genome editing (HGE). There has been an international call for public input to inform a substantive debate about benefits and risks of HGE. This study investigates the views of the sickle cell disease (SCD) community. We utilized a mixed-methods approach to

Genome editing has opened up the possibility of heritable alteration of the human germline. The potential of this powerful tool has spurred a call for establishing robust regulatory frameworks to outline permissible uses of genome editing and to map a rational and ethical course. In response, major national scientific bodies and international organizations have convened and released comprehensive reports

Gene-editing technologies have improved in ease, efficiency, and precision. Although discussions are occurring around acceptable uses of human gene editing, limited data exist on the views of genetics-trained individuals. In 2017, we distributed an anonymous online survey to assess the attitudes of members of genetics professional societies toward gene editing (N = 500). Virtually all respondents were

CRISPR-based technologies have become central to genome engineering. However, CRISPR-based editing strategies are dependent on the repair of DNA breaks via endogenous DNA repair mechanisms, which increases susceptibility to unwanted mutations. Here we complement Cas9 with a recombinase's functionality by fusing a hyperactive mutant resolvase from transposon Tn3, a member of serine recombinases, to

Genome-wide CRISPR-Cas9 essentiality screening represents a powerful approach to identify genetic vulnerabilities in cancer cells. Here, we applied this technology and designed a strategy to identify target genes that are synthetic lethal (SL) with von Hippel-Lindau (VHL) tumor suppressor gene. Inactivation of VHL has been frequently found in clear cell renal cell carcinoma. Its SL partners serve as

Through fusing CRISPR-Cas9 nickases with cytidine or adenine deaminases, a new paradigm-shifting class of genome-editing technology, termed "base editors," has recently been developed. Base editors mediate highly efficient, targeted single-base conversion without introducing double-stranded breaks. Analysis of base editing outcomes typically relies on imprecise enzymatic mismatch cleavage assays, time-consuming

There is currently no international consensus on how human germline engineering should be regulated. Existing national legislation fails to provide the governance framework necessary to regulate germline engineering in the CRISPR era. This is an obstacle to scientific and clinical advancements and inconsistent with human rights requirements. To move forward, we suggest that the human right to science

CRISPR-based editing is a revolutionary tool for genome engineering and discovery. The pace of innovation in this young field is truly astonishing, and yet significant gaps remain in the set of capabilities offered. In particular, scalable (massively parallel and multiplex) editing is only available for a small variety of edit types, placing fundamental limits on the kinds of studies researchers can

Considerable effort has been devoted to developing a comprehensive understanding of CRISPR nuclease specificity. In silico predictions and multiple genome-wide cellular and biochemical approaches have revealed a basic understanding of the Cas9 specificity profile. However, none of these approaches has delivered a model that allows accurate prediction of a CRISPR nuclease's ability to cleave a site

Nucleic acid detection is vital for agricultural applications including trait detection during breeding, pest surveillance, and pathogen identification. Here, we use a modified version of the CRISPR-based nucleic acid detection platform SHERLOCK to quantify levels of a glyphosate resistance gene in a mixture of soybeans and to detect multiple plant genes in a single reaction. SHERLOCK is rapid (∼15 min)

Prokaryotes are under nearly constant attack by viral pathogens. To protect against this threat of infection, bacteria and archaea have evolved a wide array of defense mechanisms, singly and in combination. While immune diversity in a single organism likely reduces the chance of pathogen evolutionary escape, it remains puzzling why many prokaryotes also have multiple, seemingly redundant, copies of

In the past few years, several types of artificial transcriptional activator, based on CRISPR-Cas9, have been developed and refined. Of these, in synergistic activation mediator and SunTag systems, the effector proteins, expressed in trans, can be recruited to the target sites via the MS2 RNA-binding system and GCN4-scFv antibody system, respectively. Here, we report a strong transcriptional activation

CRISPR-Cas9-Cytidine deaminase fusion enzymes-termed "base editors"-allow targeted editing of genomic deoxycytidine to deoxythymidine (C:G→T:A) without the need for double-stranded break induction. Base editors represent a paradigm shift in gene editing technology due to their unprecedented efficiency to mediate targeted, single-base conversion. However, current analysis of base editing outcomes rely

As befits an immune mechanism, CRISPR-Cas systems are highly variable with respect to Cas protein sequences, gene composition, and organization of the genomic loci. Optimal classification of CRISPR-Cas systems and rational nomenclature for CRISPR-associated genes are essential for further progress of CRISPR research. These are highly challenging tasks because of the complexity of CRISPR-Cas and their

CRISPR technology has dramatically changed scientists' ability to conduct research in medicine, biotechnology, and agriculture through faster, more efficient genome editing. A key driver of the technology's adoption is the easy, fast, and inexpensive access to vectors and the resulting next-generation tools by the nonprofit plasmid repository Addgene. Since 2013, Addgene has shipped over 100,000 CRISPR