Introduction
Clustered regularly spaced short palindromic repeats (CRISPRs) are a diverse family of adaptive immune systems used by archaea and bacteria to defend against foreign genetic elements such as plasmids and viruses.1,2,3,4CRISPR systems are extremely diverse and are currently classified into two classes, six types and dozens of subtypes.5,6For clarity, we will focus on the main themes underlying the canonical CRISPR system and the details necessary for neuroscientists to understand the molecular technologies described below. For a more complete description of the CRISPR system, an updated review should be consulted.7
The operation of a normal CRISPR system can be divided into three phases: acquisition, biogenesis and interference. In the adaptation step, small segments of DNA or RNA derived from foreign genetic elements, called spacers, are integrated into the host organism's DNA at a specific site called the CRISPR array. This is achieved through the action of a defined integration complex that includes the CRISPR-associated (Cas) proteins Cas1 and Cas2. In the biogenesis phase, the entire CRISPR array, consisting of one or more spacers interspersed with direct repeats (DR), is transcribed and then processed into short CRISPR RNAs (crRNAs) by specific host Cas enzymes or RNases. Finally, in the interference step, crRNAs direct Cas endonucleases to target sites based on sequence complementarity resulting in target cleavage and defense against the foreign genetic element. This entire process is governed by a diverse set of highly specialized Cas proteins, evolved to interact with and manipulate genetic material, which are now used to create a toolkit of molecular technologies for screening and engineering biology, as well as disease treatment.
The initial driving force behind the development of Cas proteins as a molecular technology was based on insights from the field of DNA repair, namely that DNA double-strand breaks (DSBs) stimulate endogenous DNA repair machinery and can be exploited to achieve desired gene modifications ( Figure 1A ).8,9,10,11,12,13,14While the exact mechanisms of DNA DSB repair are still being resolved,15,16,17the technologically simplistic view is that DNA DSBs are mainly repaired via non-homologous end joining (NHEJ) leading to insertions and deletions (indels) at the break site, which can lead to frameshift mutations within coding sequences. Furthermore, in the presence of a donor sequence, i.e. a single- or double-stranded DNA molecule with homology surrounding the break site and any sequence of interest in between, it can be further repaired via homology-directed repair (HDR) leading to accurate knockin mutations (Figure 1B). . The NHEJ pathway is dominant in most cell types and conditions and is active in all phases of the cell cycle, while the HDR pathway is generally less efficient and occurs only in mitotic cells during certain phases of the cell cycle.17Recently, an alternative DNA repair pathway, called alternative NHEJ (alt-NHEJ) or microhomology-mediated end joining (MMEJ), has attracted increasing attention in the field and has been exploited to induce predictable gene modifications in mitotic and postmitotic cells. .15These predictable gene alterations include short indels, single nucleotide insertions, large chromosomal deletions, and precise hits (Figures 1B-1D).18,19,20,21While these concepts were initially generated using meganucleases and designer DNA-binding proteins fused to the restriction enzyme FokI, such as zinc finger nucleases (ZFNs) and transcription activator-like nucleases (TALENs), Cas enzymes have revolutionized and democratized gene editing by providing a robust, an efficient and simple RNA-guided platform that could be widely disseminated and rationally implemented in seemingly any laboratory.
After three decades of basic life science research on CRISPR systems, the first Cas protein used for gene editing was Cas9 fromStreptococcus pyogenes, Firstin vitro22and then in eukaryotic cells.23,24,25Since these demonstrations, Cas9 has been the centerpiece of thousands of publications from laboratories around the world. We will briefly summarize the mechanism of Cas9-mediated gene editing as this knowledge provides a basis for understanding future applications as well as the advantages and disadvantages of the technology.
In nature, Cas9 is driven by double-stranded RNA, crRNA and aprev-activating crRNA (tracrRNA), to target double-stranded DNA, but in the laboratory they are combined into a single guide RNA (sgRNA) (Figure 1A).22In cells, a single Cas9 enzyme and sgRNA form a ribonucleoprotein (RNP) and scan the genome for a specific sequence motif, called a protoseparator adjacent motif (PAM).26The PAM sequence is recognized by the PAM-interacting domain of the Cas9 protein, which is used in nature to discriminate self-targeting spacers, thereby preventing autoimmunity.27The PAM sequence for SpCas9 is either "NGG" and to a lesser extent "NAG" and occurs approximately every 4-8 nucleotides in the human genome. Cas9 enzymes from other species (i.e., orthologs) share the same general mechanism but differ in primary sequence, structure, and PAM sequence requirements.5Once Cas9 finds the PAM sequence, Cas9 facilitates strand separation and the crRNA attacks the target DNA. Upon sufficient complementarity, via Watson-Crick base pairing, between the spacer sequence and the target strand, Cas9 undergoes a conformational change—placing two independent and distinct nuclease domains (ie, RuvC and HNH) close to the DNA strands leading to two individual latent DNA tracks. After generating DNA DSBs with mostly blunt ends, Cas9 holds the PAM-containing end of the break until it is removed by host enzymes.27Eventually, the DNA repair machinery takes over and repairs the DNA break, or in cases where the breaks are not repaired, the cell stops or undergoes programmed cell death.
Technically, there are many details to unpack here that have profound implications for gene editing. On the other hand, Cas9 can tolerate mismatches between sgRNA and target DNA resulting in off-target cleavage (ie, off-target effects);28,29,30,31,32and DSBs induced by Cas9 or any other endonuclease can lead to unwanted effects of DNA repair on the target other than short indels, such as large chromosomal deletions or rearrangements (ie, off-target flanking).33,34On the other hand, the incidence of off-target and on-target collateral effects is relatively low and can be detected, reduced and controlled in research settings.35On the positive side, the existence of two independent nuclease domains means that each catalytic site can be disabled individually to create single-stranded Cas9s (Cas9n) that cut only one of the two DNA strands, or together to create a 'dead' Cas9 (dCas9). enzymes that bind to DNA without cutting it.7Both of these features have been used extensively for the development of molecular technologies and will be discussed in detail below. In addition, a multitude of additional Cas9 proteins from different organisms (StCas9, NmCas9, FmCas9 and CjCas9) have been discovered that offer new possibilities, where the main research so far has been focused on discovering smaller variants that are easier to deliver or variants with different PAMs recognition sites ( 5'NNG3' for ScCas9 and 5'NNGG3' for SauriCas9) that change the targeting range of the enzyme.7With the review and discovery of new organisms, this repertoire is expected to grow even more. Cas9 has sparked a revolution in gene editing, and while drawbacks remain, the benefits have prompted a deep dive into CRISPR biology and the creation of new technologies that are changing the way biological systems are probed and programmed.
In addition to the pool of Cas enzymes provided by nature, many rationally designed or evolved Cas enzymes have been designed to introduce entirely new functions as well as to overcome inherent limitations. For example, an active area of research is focused on increasing enzyme fidelity and expanding the range of targeting (Box 1). In addition, the discovery and development of Cas9 as a gene editing tool has fueled bioinformatics efforts to mine and characterize CRISPR systems to identify other Cas enzymes with useful or unique properties. Box 2 further taps into this natural diversity by introducing new emerging gene editing technologies, including the hyper-diverse Cas12 family, the RNA-targeting Cas13 family, CRISPR-Cas transposases, and evolutionary Cas enzyme precursors.
With the discovery and development of the CRISPR-Cas enzyme, researchers have gained a powerful tool for editing and manipulating genes, genomes and cells. This has opened up a wide range of possibilities in the field of neuroscience, enabling new approaches to understanding brain development and functioning in health and disease. We then summarize various CRISPR technologies with immediate applications in the field of neuroscience, as well as emerging applications poised to answer fundamental questions in the field. For each application, we describe the technology, provide neuroscience-related examples, and highlight known shortcomings and limitations.
Unit extracts
Transcriptional modulation by dCas-based effectors
CRISPR-based technologies have advanced in the form of synthetic fusion proteins that use the programmable and RNA-driven nature of Cas enzymes to target specific genomic and transcriptional addresses. This is achieved by using catalytically inactivated Cas enzymes as general RNA-guided DNA or RNA binding proteins, which can be fused to functional protein domains or recruited in other ways (Figure 2A). The first wave of studies showed that after localization, dCas9 directly fuses with the transcriptome
Applications of CRISPR: Generating cell and organelle models
CRISPR technology was originally used to manipulate immortalized cell lines to create stable cell lines with specific gene knockouts and knock-ins that introduce precise genetic mutations or fluorescent tags to proteins of interest. However, neuroscientists prefer models that better represent the complexity of brain cell types, including progenitor cells, stem cells induced into neuronal cell types, and 2D or 3D models based on differentiated stem cells. CRISPR technology has made it easier
CRISPR application: Generating animal models by editing zygotes
One of the firstliveThe application of Cas9 was to create genetically modified model (GEM) organisms by introducing SpCas9 and sgRNA into single-cell zygotes.101The principle here is that gene modifications are created in single cells, which then develop into GEM founder organisms (Figure 3). Compared to previous approaches based on modification and selection of ES cellsin vitro, zygote processing offers several key advantages. First, gene editing in zygotes drastically shortens the time
Application of CRISPR:livegene editing in animal models
Instead of creating and maintaining GEM organisms, CRISPR offers the ability to directly examine genetic elementslivein live animals—disrupting genetic elements in relevant cells and tissues and directly performing phenotyping experiments (Figure 4). This has the advantage of avoiding the need to create and maintain GEM organizations, as well as providing plenty of flexibility in terms of when and where disruption occurs. Ultimately, it speeds up the pace drastically
Emerging application: mobile scheduling
Many biological functions require the orchestrated regulation of complex gene networks to achieve a desired phenotype or function (Figure 5). Before the development of CRISPR as a gene editing technology, multiplex gene editing was possible, but cumbersome and limited because each target of interest required a separate effector protein (eg, ZFN and TALEN).167On the other hand, CRISPR systems are uniquely suited to multiplexing, where in nature CRISPR arrays encode a multiplex
New possibilities at the intersection of neuroscience and future genetics
The advent of omics datasets related to human neurological diseases has generated a large number of candidate genes whose contribution to the disease needed to be evaluated.179,180,181The contribution of each gene in the genome to the phenotype can be functionally assessed using forward genetic screens. Advances in deep sequencing, DNA synthesis, and programmable RNA-guided disruption technologies such as RNAi and CRISPR have fueled progress in this field by providing (1) complete genome sequences
Application of CRISPR: Cell Lineage Tracing and Molecular Imaging
Most methods in the life sciences rely on disruptive experiments in which cells or tissues are disrupted and/or fixed, and components are measured—giving a rich picture of the system at a given point in time, but providing little or no information about past biological events. While live cell imaging enables the monitoring of cellular systems over time, these methods do not go beyond a narrow field of view or whole-organism level. Enabling technologies
Conclusions and perspectives
In this initial section, we wanted to provide a basis for understanding the development and application of CRISPR-Cas technologies, their current limitations and future prospects in neuroscience and the wider biomedical sciences. The field of genome engineering continues to advance at a rapid pace, and new tools and approaches for understanding and engineering biology are constantly emerging. We are beginning to see hints of molecular technologies that will complement or replace CRISPR
Thank you
We would like to acknowledge the valuable contributions of Dr. Katherine Guzzetta, Dr. Yingjun Liu, Dr. Antonio Jose da Silva Santinha, and Dr. Alessio Strano who provided comments on this manuscript. We also express our gratitude to members of the Platt laboratory for valuable scientific discussions. This work was supported byEuropean Research Councilapprove851021;Botnar Child Health Research Center Multi-Investigator ProjectiFast Track call grants;National centers
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