Nobel Prize in Chemistry 2020

Emmanuelle Charpentier and Jennifer Doudna share the award for developing the precise genome-editing technology.

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.

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.

CRISPR/Cas is a microbial immune system that is known to protect bacteria from virus infection. These authors show that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA.

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 possessing CRISPR-type immunity encode Cas proteins. This study now describes an alternative pathway in Streptococcus pyogenes that employs trans-encoded small RNA that directs the processing of precursor RNA into crRNAs through endogenous RNase III and the CRISPR-associated Csn1 protein.

The genome of human cells is edited using the bacterial RNA-guided Cas9 endonuclease.

A CRISPR-Cas system is harnessed to introduce template-driven mutations in S. pneumoniae and E. coli at high efficiency without requiring selectable markers.

RNA polymerase III–driven single guide RNA and a germ line promoter–driven expression of Cas9 enzyme allow heritable, targeted genome modifications in Caenorhabditis elegans.

CAS9 nucleases, a new tool for genome editing, show significant offtarget activity.

Analyses of the determinants of the specificity of Cas9 nuclease provide rules for selecting optimal target sites.

This Perspective describes current and prospective advances in genome engineering made possible with the CRISPR-Cas9 system.

This study defines how a short DNA sequence, known as the PAM, is critical for target DNA interrogation by the CRISPR-associated enzyme Cas9 — DNA melting and heteroduplex formation initiate near the PAM and extend directionally through the remaining target sequence, and the PAM is also required to activate the catalytic activity of Cas9.

Crystal structure of the RNA-guided endonuclease Cas9 bound to a guide RNA and a target DNA duplex reveals how base-specific recognition of a short motif known as 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.

In the presence of a short DNA oligonucleotide containing a protospacer adjacent motif, a guide-RNA-programmed Cas9 is able to specifically bind and/or cleave single-stranded RNA—this system can be used to isolate specific endogenous RNA transcripts from a cell lysate without any tag or modification.

The CRISPR-associated protein Cpf1 from Francisella novicida is a novel enzyme with specific, dual-endoribonuclease–endonuclease activities in precursor crRNA processing and crRNA-programmable cleavage of target DNA.

CRISPR/Cas9 DNA editing creates a double-stranded break in the target DNA, which can frequently generate random insertion or deletion of bases (indels); a new genome editing approach combining Cas9 with a cytidine deaminase is described here, which corrects point mutations more efficiently than canonical Cas9, while avoiding double-stranded breaks and indel formation.

A new DNA-editing technique called prime editing offers improved versatility and efficiency with reduced byproducts compared with existing techniques, and shows potential for correcting disease-associated mutations.

Cas1–Cas2 selects precursor prespacers from DNA fragments in a length- and PAM-sequence-dependent manner, and these precursors are trimmed by DnaQ exonucleases to enable integration into the CRISPR locus in the correct orientation.

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.

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.

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

The ultimate goal of genome editing is to be able to make any specific change to the blueprint of life. A ‘search-and-replace’ method for genome editing takes us a giant leap closer to this ambitious goal.

Clinical trials of genome-editing agents — including CRISPR–Cas9 editors, zinc finger nucleases and TALENs — are pushing ex vivo, immuno-oncology and in vivo treatment frontiers.

Tuning CRISPR–Cas9 nuclease specificity enables precision genome engineering. Identifying arginine residues along the bridge helix of SpCas9 that mediate Cas9 mismatch sensitivity enabled engineering of Cas9 with increased specificity in human cells.

Type III CRISPR-Cas systems are able to target transcriptionally active DNA sequences in phages and plasmids. Here, the authors reveal the mechanism of the target nucleic acid preference of Type III-A CRISPR-Cas complexes at the transcription bubble by a combination of structural and biochemical approaches.

Doudna and colleagues determine the mechanisms used by type V anti-CRISPR proteins. AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the Cas12a-bound guide RNA, while AcrVA4 and AcrVA5 inhibit recognition of dsDNA.

CRISPR–CasX represents a distinct RNA-guided platform that is functionally separate from Cas9 and Cas12a and is active for bacterial and human genome modification.

In this protocol, the authors describe how to design, synthesize, and deliver CRISPR–Cas9 RNPs to primary CD4⁺ T cells for targeted gene knockout. They then show how the edited cells can be used for the analysis of host factors in HIV replication.

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.

Gold nanoparticles carrying Cas9 ribonucleoprotein and donor DNA, and complexed with endosomal disruptive polymers, correct the DNA mutation that causes Duchenne muscular dystrophy in mice, with minimal off-target effects.

A new engineered version of SpCas9, called HypaCas9, displays enhanced accuracy of editing without significant loss of efficiency at the desired target.

Crystal structures of unprocessed and mature crRNA-bound LbaCas13a shed light upon catalytic residues involved in crRNA maturation and mechanisms blocking Cas13a nuclease activity before target-RNA binding.

Using a metagenomic approach, three types of CRISPR–Cas systems have been discovered in uncultivated bacterial and archaeal hosts from a variety of different environments.

The CRISPR-associated bacterial enzyme C2c2 is shown to contain two separable, distinct sites for the highly sensitive detection and cleavage of single-stranded RNA.

CRISPR-Cas9 has enabled an unprecedented ability to manipulate the genome yet it is still poorly understood how target recognition functions at a molecular level. Here the authors use single-molecule FRET to probe Cas9-target interaction and identify distinct search and proofreading states.

Cas1–Cas2 integrase achieves full-site integration only for proper targets and protospacers, whereas at non-CRISPR sites, integration stalls at the half-site intermediate, thereby protecting host genome integrity during CRISPR immune adaptation.

A fluorescence-based approach defines new features of Cas9 that control the specificity of RNA-guided DNA cleavage in CRISPR genome editing technology.

The structure of the Cas1–Cas2 complex bound to a protospacer sequence illustrates how foreign DNA is captured and measured by bacterial proteins in preparation for integration into CRISPR loci.

The bacterial CRISPR/Cas system acquires short phage sequences known as spacers that integrate between CRISPR repeats and constitute a record of phage infection; this study shows that the Cas1–Cas2 complex is the minimal machinery required for spacer acquisition and the complex integrates oligonucleotide DNA substrates into acceptor DNA in a manner similar to retroviral integrases and DNA transposases with Cas 1 as the catalytic subunit and Cas2 acting to increase integration activity.

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.

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.

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 possessing CRISPR-type immunity encode Cas proteins. This study now describes an alternative pathway in Streptococcus pyogenes that employs trans-encoded small RNA that directs the processing of precursor RNA into crRNAs through endogenous RNase III and the CRISPR-associated Csn1 protein.

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.

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.

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.

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.

RNA-targeting CRISPR. In this Perspective, Smargon, Shi and Yeo discuss the rapid development of the RNA-targeting CRISPR–Cas engineering system and highlight how this can be leveraged to further understand RNA biology.

Harnessing DNA repair pathways in genome editing In this Review, Yeh, Richardson and Corn discuss the DNA repair pathways that underlie genome editing and recent improvements and strategies to yield desired genomic alterations in cells and organisms.

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.

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.

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.

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.

In this Review, Wiedenheft and colleagues retrace events that led from early endeavours to understand the role of Cas9 in CRISPR-mediated adaptive immunity to current efforts aimed at developing this enzyme for programmable genetic editing.

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.

CRISPR–Cas genome editing and next-generation sequencing are driving advances in cancer modelling and functional cancer genomics. Their application to autochthonous mouse models of human cancer to generate and analyse multiplexed and/or combinatorial alterations in vivo is reviewed here.

The application of precision gene editing has great potential to accelerate basic research and advance clinical practice in nephrology. Here, the authors discuss this technology and the challenges and potential of genome editing in the kidney.

This Perspective explores the diversity, mechanisms and practical aspects of natural and engineered CRISPR-associated nucleases for genome engineering applications.

CRISPR has rapidly become an indispensable tool for biological research. Here Mazhar Adli reviews the current toolbox for editing and manipulating the genome and looks toward future developments in this fast moving field.

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.

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.

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.

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.

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.

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.

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.

The CRISPR system uses small RNAs to provide bacteria and archaea with an adaptive defence mechanism against foreign nucleic acids. Recent studies have increased the understanding of mechanisms of CRISPR interference, its roles in microbial physiology and evolution and its potential applications.

Pigs can be genetically modified to inactivate endogenous retroviruses and to display enhanced compatibility with the human immune system using a combination of CRISPR–Cas9 and transposon technologies.

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.

Rice genes are replaced using chemically modified donor DNA and CRISPR targeting.

CRISPR technology is emerging as a potential antimicrobial against antimicrobial-resistant bacteria. Here the authors develop a bacteriophage delivered Cas13a system for killing target bacteria and detecting bacterial genes.

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.

Domain insertion into the loop region of AcrIIC1 leads to a variant with enhanced inhibitory activity toward Neisseria meningitides Cas9, while structure-guided design turns AcrIIC1 into a potent inhibitor of Staphylococcus aureus Cas9.

A continuously evolved adenine base editor is compatible with various Cas proteins and mediates efficient A•T-to-G•C base conversions at a wide variety of PAM sites.

Methods to induce edited somatic plant cells to form meristems circumvent tissue culture and enable genome editing of a wider set of plant species.

Polymer nanocapsules, assembled around a ribonucleoprotein complex of Cas9 nuclease and a single-guide RNA, enable safe and efficient gene editing in vitro and in vivo.

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.

Split Cpf1 pairs are identified to enable chemical- and light-induced genome editing via dimerization. Another pair of split Cpf1 can be used to activate gene expression with high efficiency in cells and in mice.

Orthologous therapeutic proteins can elude the adaptive immune system and allow for effective repeated dosing, as shown for CRISPR gene editing with orthologues of adeno-associated viral capsids and of the Cas9 protein.

A wild tomato species is domesticated by targeted CRISPR–Cas9 editing of just six genes.

Some phages carry genes coding for anti-CRISPR (Acr) proteins that interfere with the activity of bacterial CRISPR-Cas systems. Here, Hynes et al. characterize a new Acr family from streptococcal phages and investigate its potential in genome-editing applications.

Gene editing of a single gene in the brain of an adult mouse model of fragile X syndrome, achieved via the intracranial injection of a nonviral Cas9 delivery vehicle, rescues mice from the exaggerated repetitive behaviours caused by the disease.

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.

Gold nanoparticles that passively deliver CRISPR machinery to blood repopulating cells have been developed and are shown to be capable of precise editing of multiple genetic loci of therapeutic interest without cytotoxicity or reduced fitness.

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 dSaCas9^KRAB to the cholesterol regulator Pcsk9, and show repression up to 24 weeks and reduced cholesterol levels in mice.

Partial substitution of CRISPR RNAs with DNA nucleotides retains CRISPR–Cas9 genome editing activity while enhancing efficiency and specificity within cells, suggesting that DNA–RNA hybrids may be economical reagents for targeted genome editing.

A cloud-based machine-learning software that scores individual guide–target pairs and provides an overall summary score for a given guide that outperforms competing algorithms for the prediction of CRISPR–Cas9 off-target effects.

The expression of two specific DNA-repair factors promotes homology-directed repair and enhances the precision of CRISPR–Cas9 gene editing at multiple loci in human cells, including patient-derived induced pluripotent stem cells.

Retinitis pigmentosa is mainly caused by mutations that initially affect survival of rod photoreceptors, leading to secondary loss of cones. Here the authors use gene editing to prevent rod degeneration, leading to survival of cones and improved vision in mice.

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.

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.

CRISPR-Cas9-mediated genome editing corrects a hereditary tyrosinemia disease mutation in the liver of adult mice.