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Nobel Prize in Chemistry 2020

The 2020 Nobel Prize in Chemistry has been awarded to Emmanuelle Charpentier and Jennifer Doudna for their pioneering work in gene-editing. To celebrate the award, Nature Research presents this Collection including publications from the prize winners, and further research and review content focused on CRISPR-Cas, and beyond — work that is opening up countless new and exciting avenues of research and application across biology.

Featured Content

A bacterial enzyme that uses guide RNA molecules to target DNA for cleavage has been adopted as a programmable tool to site-specifically modify genomes of cells and organisms, from bacteria and human cells to whole zebrafish.

News & Views | | Nature

CRISPR–Cas gene editors are now both moving into the clinic and being embraced as a means to find and validate drug targets. But for Jennifer Doudna, who helped pioneer this promise with her work at UC Berkeley, the full potential of these tools will only be unleashed when they can be used at scale. To this end, Doudna and colleagues partnered last year with GlaxoSmithKline to launch the Laboratory for Genomic Research (LGR), a US$67 million effort aimed at industrializing the CRISPR–Cas workflow for the detailed exploration of human genetics. One year on, she spoke with Asher Mullard about her hopes for CRISPR–Cas editors as drug discovery tools, the types of projects the LGR is working on and the challenges they face.

An Audience With | | Nature Reviews Drug Discovery

CRISPR/Cas is a microbial immune system that is known to protect bacteria from viral infection. It is now shown that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA of both viral and plasmid origin. The system seems remarkably adapted to this end, and it is thought that CRISPR/Cas could be used to naturally generate safer and more robust bacteria that are resistant to the acquisition and spread of antibiotic resistance genes.

Article | | Nature

CRISPR is a microbial RNA-based immune system protecting against viral and plasmid invasions. The CRISPR system is thought to rely on cleavage of a precursor RNA transcript by Cas endonucleases, but not all species with CRISPR-type immunity encode Cas proteins. A new study reveals an alternative pathway for CRISPR activation in the human pathogen Streptococcus pyogenes, in which a trans-encoded small RNA directs processing of precursor RNA into crRNAs through endogenous RNase III and the CRISPR-associated Csn1 protein.

Article | | Nature

CRISPR/Cas9-based DNA targeting has quickly become a leading tool in the fields of synthetic biology and genome engineering. It exploits the ability of a bacterial endonuclease, Cas9, guided by an RNA molecule, to target virtually any matching DNA sequence of interest for binding and/or cleavage.

This paper reports the use of single-molecule and bulk biochemical experiments to reveal the mechanism by which RNA-guided Cas9 locates unique 20-base-pair sequences within DNA genomes, which can be billions of base pairs long. The results highlight the role of a trinucleotide protospacer adjacent motif (PAM) in recruiting Cas9–RNA complexes to potential DNA target sites, and in catalytically activating the nuclease. Target DNA sequences are recognized via a 'zip-up' mechanism, where the sequential formation of RNA–DNA base pairs offsets the energetic cost of unwinding the DNA double helix. In addition to its relevance for gene manipulation, this work reveals how DNA is interrogated by Cas9–RNA in its role as an effector of adaptive immunity in bacteria.

Article | | Nature

The bacterial Cas9 nuclease is an RNA-guided DNA endonuclease found in bacterial CRISPR defence systems and is also widely used as a genetic engineering tool. Cas9 associates with a guide RNA, forms a complex with complementary duplex DNA, and cleaves the DNA. For cleavage to occur, the DNA target must contain a trinucleotide motif known as PAM. Martin Jinek and colleagues have solved the structure of Cas9 bound to a guide RNA and a PAM-containing duplex DNA. The structure reveals how base-specific recognition of PAM in the DNA target results in localized strand separation in the DNA immediately upstream of the PAM, allowing the target DNA strand to hybridize to the guide RNA.

Letter | | Nature

The bacterial CRISPR immune defence system, and its effector Cas9 in particular, have recently been exploited for sequence-specific genome editing in eukaryotic cells. Cas9 binds a guide RNA and in the presence of a DNA motif known as protospacer adjacent motif (PAM), is able to cleave the target DNA. New work by Jennifer Doudna and colleagues reveals the unexpected result that in the presence of a DNA oligomer containing PAM, a guide RNA-programmed Cas9 is able to cleave single-stranded RNA as well. They show that this system can also be used to isolate specific endogenous RNA transcripts, without any tag or modification, from a cell lysate. Thus, the system can be programmed to either bind or cut desired RNA targets, depending on the PAM used. This work and points the way towards possible new technologies for programmable RNA recognition.

Letter | | Nature

The bacterial immune system, CRISPR, utilizes a small RNA guide, or crRNA, to target a nucleolytic CRISPR complex to DNA with a complementary sequence. This process has been widely exploited for various types of genome engineering. Previously described CRISPR systems utilize one nuclease, such as Cas6, to generate the mature crRNA, and a second, such as Cas9, to cleave the target DNA. Two studies illustrate a different approach that involves the Cpf1 protein. Emmanuelle Charpentier and colleagues report that type V-A Cpf1 protein from Francisella novicida functions as a minimalistic CRISPR system. It is a dual-nuclease enzyme that can perform both the pre-crRNA processing and DNA cleavage activities, having distinct active domains for the two substrates. Zhiwei Huang and colleagues solve the crystal structure of monomeric Lachnospiraceae bacterium Cpf1 protein bound to crRNA, showing how binding induces conformational changes in the nuclease.

Letter | | Nature

The CRISPR/Cas technology widely used for genome editing involves formation of a double-strand break in the target DNA sequence. When used to modify a single nucleotide, this procedure frequently generates DNA insertions or deletions (indels). David Liu and colleagues describe an approach that obviates DNA cleavage, as a means to avoid such off-target mutations. This 'base editing' method, which utilizes a composite enzyme consisting of CRISPR/Cas9 and the APOBEC1 deaminase, can directly convert C to T (or G to A). They also describe modifications that increase the yield of the desired correction and significantly suppressing indel formation.

Letter | | Nature

The scientific, technical and ethical aspects of using CRISPR technology for therapeutic applications in humans are discussed, highlighting both opportunities and challenges of this technology to treat, cure and prevent genetic disease.

Review Article | | Nature

Genomic analyses of major clades of huge phages sampled from across Earth’s ecosystems show that they have diverse genetic inventories, including a variety of CRISPR–Cas systems and translation-relevant genes.

Article | Open Access | | Nature

Eric Lander, Françoise Baylis, Feng Zhang, Emmanuelle Charpentier, Paul Berg and specialists from seven countries call for an international governance framework.

Comment | | Nature

From the Winners

While current CRISPR-Cas9 tools have revolutionized genome editing, they are not suitable for applications at elevated temperatures. Here, the authors characterize GeoCas9 from Geobacillus stearothermophilus, which is active up to 70°C and is stable in human plasma.

Article | Open Access | | Nature Communications

One of the main concerns about the use of CRISPR in genomic editing is the possibility of 'off-target' events. Scientists have been modifying the central enzyme involved in CRISPR editing, Cas9 or its homologues, to reduce this unwanted property. Jennifer Doudna and colleagues describe a new version of this nuclease, HypaCas9, which enables more accurate editing, without substantial loss of efficiency on the desired target.

Letter | | Nature

Current CRISPR–Cas technology is based on systems identified from cultured bacteria, whereas the enzymes from the numerous prokaryotes that have not been cultured have remained unexplored. By using cultivation-independent genome-resolved metagenomics, Jillian Banfield, Jennifer Doudna and colleagues identify and then functionally characterize new CRISPR–Cas systems. These include the first reported Cas9 in the archaeal domain of life, which was thought to lack such systems, as well as compact CRISPR–CasX and CRISPR–CasY systems. Genomic exploration of environmental microbial communities gives access to unprecedented genome diversity with the potential to revolutionize microbe-based biotechnologies.

Letter | | Nature

The programmed sequence-specific cleavage of RNA and DNA by CRISPR-associated enzymes has revolutionized genome editing. An alternative to canonical Cas9 nuclease, C2c2, was recently described. Jennifer Doudna and colleagues have probed the biochemistry of this enzyme further, and find that it contains two separable distinct sites that catalyse RNA cleavage. The authors exploit the properties of the second site to show that the enzyme can be used for highly sensitive detection and cleavage of single-stranded RNA.

Letter | | Nature

The CRISPR genome editing technology now used widely in mutant analysis in a variety of organisms is only a few years old, and the technology is still being optimized to enhance its specificity and efficiency. The central catalytic activity, RNA-guided cleavage that is directed against the genomic DNA, is carried out by the Cas9 endonuclease. Jennifer Doudna and colleagues use a fluorescence-based approach to define new features of Cas9 that control the specificity of RNA-guided DNA cleavage in CRISPR genome-editing technology.

Letter | | Nature

The CRISPR–Cas system widely used for genome editing in a variety of organisms derives from a bacterial immune system. In bacteria, DNA sequences from invading phage are incorporated into the host genome in loci known as CRISPR. How these 'spacer' sequences of 30–40 base pairs are selected and generated was unclear. Jennifer Doudna and colleagues now describe the structure of the Cas1–Cas2 complex bound to a protospacer sequence. The structure illustrates how the foreign DNA is captured and cleaved by the host proteins in preparation for integration into CRISPR loci.

Letter | | Nature

The once fanciful idea that bacteria might have immunological memory became accepted fact with the discovery that the CRISPR–Cas gene loci evolve rapidly to acquire short phage sequences, or spacers, which then integrate between CRISPR repeats and constitute a record of phage infection. These spacers are transcribed into small CRISPR RNAs (crRNAs) that are used to target the DNA of invading viruses. Two papers published in this issue of Nature describe molecular details about how bacteria create a DNA memory of the invading virus. Jennifer Doudna and colleagues show that the purified Escherichia coli Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA in a manner similar to retroviral integrases and DNA transposases. Cas1 is the catalytic subunit, while Cas2 increases integration activity; together they form the minimal machinery required for spacer acquisition. Luciano Marraffini and colleagues show that in the type II CRISPR–Cas system of Streptococcus pyogenes, the Cas9 nuclease that inactivates invading viral DNA using the crRNA as a guide is also required for the incorporation of new spacer sequences, by a yet to be determined mechanism.

Article | | Nature

The CRISPR–Cas system mediates immunity to foreign DNA sequences that are integrated as spacers between repeats in the CRISPR locus. Work from Doudna and colleagues shows that nucleases Cas1 and Cas2 form a stable complex that recognizes the CRISPR leader-repeat sequence, thus determining the site of integration.

Article | | Nature Structural & Molecular Biology

Bacterial cells use CRISPRs (clustered regularly interspaced short palindromic repeats) to defend against invading phages. The central catalytic component in this process is Cascade, a 12-subunit complex consisting of proteins and RNA. The structure of Cascade, free and bound to target RNA, has now been solved by cryoelectron microscopy and three-dimensional reconstruction. These structures show the changes in architecture that are induced by target binding, and will assist future studies addressing how these conformational changes affect restriction of the phage.

Letter | | Nature

In bacteria and archaea, clustered regularly interspaced short palindromic repeat (CRISPR) loci are unique pieces of foreign DNA (spacers) separated by repetitive sequences specific to the host. They provide an adaptive immune system against bacteriophages and plasmids. CRISPR loci are transcribed and processed by three endonucleases to produce short RNAs (crRNAs). Structures of Cse3-type endonuclease bound to its cognate repetitive RNA shows an RNA-induced conformational change in enzyme that aligns the RNA for site-specific cleavage.

Article | | Nature Structural & Molecular Biology

CRISPR is a microbial RNA-based immune system protecting against viral and plasmid invasions. The CRISPR system is thought to rely on cleavage of a precursor RNA transcript by Cas endonucleases, but not all species with CRISPR-type immunity encode Cas proteins. A new study reveals an alternative pathway for CRISPR activation in the human pathogen Streptococcus pyogenes, in which a trans-encoded small RNA directs processing of precursor RNA into crRNAs through endogenous RNase III and the CRISPR-associated Csn1 protein.

Article | | Nature

Reviews

The newest CRISPR–Cas genome editing technologies enable precise and simplified formation of crops with increased yield, quality, disease resistance and herbicide resistance, as well as accelerated domestication. Recent breakthroughs in CRISPR–Cas plant biotechnologies improve reagent delivery, gene regulation, multiplexed gene editing and directed evolution.

Review Article | | Nature Reviews Molecular Cell Biology

The causal role of chromatin modifications has been difficult to study in the brains of behaving animals. Yim, Teague and Nestler review locus-specific neuroepigenome-editing tools to define causal relationships between chromatin modifications and their molecular, cellular, circuit and behavioural consequences.

Review Article | | Nature Reviews Neuroscience

The search for effective therapies for neurological disease has been impeded by our limited understanding of the causative molecular and cellular mechanisms. Kampmann describes how new CRISPR-based functional genomics approaches can uncover disease mechanisms and therapeutic targets in neurological diseases.

Review Article | | Nature Reviews Neurology

The scientific, technical and ethical aspects of using CRISPR technology for therapeutic applications in humans are discussed, highlighting both opportunities and challenges of this technology to treat, cure and prevent genetic disease.

Review Article | | Nature

In this Perspective, Lea and Niakan describe advances in CRISPR/Cas9 genome editing techniques and discuss ethical questions and potential clinical implications of this technology.

Perspective | | Nature Cell Biology

In vivo genome editing requires delivery systems that are efficient, safe and tissue specific. This Review outlines the materials and delivery strategies currently used, and the challenges and potential solutions in in vivo genome editing, aiming to stimulate further development of engineered materials for in vivo delivery of genome-editing machinery.

Review Article | | Nature Reviews Materials

CRISPR–Cas systems have revolutionized genome editing, and the CRISPR–Cas toolkit has been expanding to include single-base editing enzymes, targeting RNA and fusing inactive Cas proteins to effectors that regulate various nuclear processes. Consequently, CRISPR–Cas systems are being tested for gene and cell therapies.

Review Article | | Nature Reviews Molecular Cell Biology

CRISPR systems have enabled important advances in cancer research by accelerating the development of study models or as a tool in genetic screening studies to discover and validate therapeutic targets. The authors of this Review discuss these applications and new potential uses, such as cancer detection and development of anticancer therapies.

Review Article | | Nature Reviews Clinical Oncology

Genome editing through direct editing of bases holds promise for achieving precise genomic changes at single-nucleotide resolution while minimizing the occurrence of potentially mutagenic double-strand DNA breaks. In this Review, Rees and Liu provide a comprehensive account of the state of the art of base editing of DNA and RNA, including the progressive improvements to methodologies, understanding and avoiding unintended edits, cellular and organismal delivery of editing reagents and diverse applications in research and therapeutic settings.

Review Article | | Nature Reviews Genetics

The rapid development of CRISPR-based gene manipulation has enabled various approaches for high-throughput functional genomics. This Review guides users through the practicalities of CRISPR-based functional genomics screens, including study design options, best-practice approaches, pitfalls to avoid and data analysis strategies.

Review Article | | Nature Reviews Genetics

Owing to their programmable ability to cut specific nucleic acid sequences, CRISPR–Cas systems have been used for precise genome engineering. In this Review, the authors discuss the chemistry and molecular mechanisms of interference by single-effector CRISPR–Cas proteins.

Review Article | | Nature Reviews Chemistry

Insights into eukaryotic, bacterial and archaeal RNA-based regulatory systems, including microRNAs, small interfering RNAs, clustered regularly interspaced short palindromic repeats (CRISPR) RNA and small RNAs that are dependent on the RNA chaperone protein Hfq, have revealed that they achieve specificity using similar strategies. Specifically, the presentation of short 'seed sequences' within a ribonucleoprotein complex facilitates the search for and recognition of targets.

Review Article | | Nature Reviews Molecular Cell Biology

The use of CRISPR–Cas technology for gene editing has rapidly become widespread. Here, Corn and colleagues discuss the applications of this revolutionary tool in drug discovery and development, describing how it could make substantial contributions to target identification and validation, animal models and cell-based therapies.

Review Article | | Nature Reviews Drug Discovery

Applying CRISPR–Cas9 genome editing technologies in safe and reliable ways requires a firm appreciation of the specificity of target-site recognition and cleavage. In this Review the authors discuss various approaches for characterizing off-target effects of CRISPR–Cas9 genome editing, how mechanistic knowledge can drive the engineering of more-specific nucleases, and the implications for research and therapeutic applications.

Review Article | | Nature Reviews Genetics

The CRISPR–Cas9 (clustered regularly interspaced short palindromic repeats–CRISPR-associated 9) system provides many avenues for improving how we generate models of cancer. This system has numerous uses, including providing a means to understand the importance of genetic alterations as a tumour evolves, and CRISPR–Cas9 may potentially constitute a therapeutic strategy in the future.

Progress | | Nature Reviews Cancer

The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems are immunity systems that are present in many bacteria and archaea. Here, Koonin and colleagues present a new classification of these systems and introduce a new nomenclature of the genes in the CRISPR–casloci that better reflects the relationships between the proteins.

Opinion | | Nature Reviews Microbiology

Research articles

CRISPR–Cas9 systems have revolutionized the field of genome editing. This work reports rare structures of a Cas9 enzyme (St1Cas9) in its HNH catalytic state, providing mechanistic insights related to DNA recognition and cleavage, and structure-guided engineering is used for expansion of the PAM recognition.

Article | | Nature Catalysis

CRISPR-based nucleic acid detection is used in a platform that can simultaneously detect 169 human-associated viruses in multiple samples, providing scalable, multiplexed pathogen detection aimed at routine surveillance for public health.

Article | Open Access | | Nature

Bacteria use CRISPR-Cas systems to protect themselves against viral infections. Here, Watson et al. show that a type I CRISPR-Cas system can induce abortive viral infection, where infected cells do not survive but viral propagation is decreased, thus protecting the bacterial population.

Article | Open Access | | Nature Communications

Eric Aird et al. present a strategy to increase the efficiency of homology-directed repair in CRISPR/Cas9-mediated genome editing. They show that tethering a single-stranded oligodeoxynucleotide to the ribonucleoprotein complex using a fused HUH endonuclease increases editing efficiency by up to 30-fold.

Article | Open Access | | Communications Biology

Repression of gene transcription using CRISPR-Cas9 has been achieved in vitro but not for delivery into adult animal models. Here, the authors use AAV8 to deliver the transcriptional repressor dSaCas9KRAB to the cholesterol regulator Pcsk9, and show repression up to 24 weeks and reduced cholesterol levels in mice.

Article | Open Access | | Nature Communications

CRISPR/Cas9-mediated gene editing is an emerging strategy to treat Duchenne muscular dystrophy. Here the authors develop multiple CRISPR/Cas9-based approaches to correct different dystrophin gene mutations, and show significant restoration of dystrophin expression in skeletal and cardiac muscle in mice.

Article | Open Access | | Nature Communications

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene. Here, Ousterout et al. use multiplexed CRISPR/Cas9 genome editing to excise a large portion of the gene that carries over 60% of known dystrophin mutations. They show that this excision restores dystrophin expression in patient-derived cells.

Article | | Nature Communications