The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.DOI: http://dx.doi.org/10.7554/eLife.04766.001
Mammalian neural stem cells (NSCs) have the capacity to both self-renew and to generate all the neuronal and glial cell-types of the adult nervous system. Global chromatin changes accompany the transition from proliferating NSCs to committed neuronal lineages, but the mechanisms involved have been unclear. Using a proteomics approach, we show that a switch in subunit composition of neural, ATP-dependent SWI/SNF-like chromatin remodeling complexes accompanies this developmental transition. Proliferating neural stem and progenitor cells express complexes in which BAF45a, a Krüppel/PHD domain protein and the actin-related protein BAF53a are quantitatively associated with the SWI2/SNF2-like ATPases, Brg and Brm. As neural progenitors exit the cell cycle, these subunits are replaced by the homologous BAF45b, BAF45c, and BAF53b. BAF45a/53a subunits are necessary and sufficient for neural progenitor proliferation. Preventing the subunit switch impairs neuronal differentiation, indicating that this molecular event is essential for the transition from neural stem/progenitors to postmitotic neurons. More broadly, these studies suggest that SWI/SNF-like complexes in vertebrates achieve biological specificity by combinatorial assembly of their subunits.
Mammalian SWI/SNF [also called BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodeling complexes are essential for formation of the totipotent and pluripotent cells of the early embryo. In addition, subunits of this complex have been recovered in screens for genes required for nuclear reprogramming in Xenopus and mouse embryonic stem cell (ES) morphology. However, the mechanism underlying the roles of these complexes is unclear. Here, we show that BAF complexes are required for the self-renewal and pluripotency of mouse ES cells but not for the proliferation of fibroblasts or other cells. Proteomic studies reveal that ES cells express distinctive complexes (esBAF) defined by the presence of Brg (Brahma-related gene), BAF155, and BAF60A, and the absence of Brm (Brahma), BAF170, and BAF60C. We show that this specialized subunit composition is required for ES cell maintenance and pluripotency. Our proteomic analysis also reveals that esBAF complexes interact directly with key regulators of pluripotency, suggesting that esBAF complexes are specialized to interact with ES cell-specific regulators, providing a potential explanation for the requirement of BAF complexes in pluripotency.BAF complexes ͉ BAF155 ͉ Brg E S cells are pluripotent cells capable of both limitless selfrenewal and differentiation into all embryonic lineages. These abilities are conferred by various mechanisms, including transcription factors (1-3), possibly Polycomb complexes (4, 5), microRNAs (6), and histone modification enzymes (7) that work in coordination to maintain the expression of pluripotency genes while repressing lineage-determinant genes. The involvement of such mechanisms in pluripotency has been investigated extensively in recent years (reviewed in ref. 8), but the role of chromatin remodeling enzymes remains unclear.The mammalian genome encodes about 30 SWI2/SNF2-like ATPases, which are assembled into SWI/SNF-like complexes with ATP-dependent chromatin remodeling activity. Of these, Brg and Brm are alternative ATPases of a family of 2-MDa multisubunit SWI/SNF or BAF complexes and make up the prototypic mammalian SWI/SNF-like chromatin remodeling complexes (9, 10). BAF complexes have been shown to be essential for many aspects of mammalian development (11-13). A role of BAF complexes in pluripotency is suggested by observations that deletion of Brg, BAF155 (or Srg3), and BAF47 (or hSNF5) all lead to peri-implantation lethality and failure of the totipotent cells that give rise to both the inner cell mass and trophoblast to survive and grow (14-16). The catalytic ATPase subunit, Brg, also was recovered in screens for factors essential for nuclear reprogramming (17) and to ES cell morphology (18). In addition, ES cells lacking BAF250 have defects in ES cell maintenance and differentiation (19,20). However, the mechanism by which BAF complexes help to establish and maintain pluripotency is not understood.In vitro, BAF complexes use energy generated from ATP hydrolysis to alter DNA-nucleosome contacts (21) and can also e...
One of the most distinctive steps in the development of the vertebrate nervous system occurs at mitotic exit when cells lose multi-potency and begin to develop stable connections that will persist for a lifetime 1,2 . This transition is accompanied by a switch in ATP-dependent chromatinremodelling mechanisms that appears to coincide with the final mitotic division of neurons. This switch involves the exchange of the BAF53a (also known as ACTL6a) and BAF45a (PHF10) subunits within Swi/Snf-like neural-progenitor-specific BAF (npBAF) complexes for the homologous BAF53b (ACTL6b) and BAF45b (DPF1) subunits within neuron-specific BAF (nBAF) complexes in post-mitotic neurons. The subunits of the npBAF complex are essential for neural-progenitor proliferation, and mice with reduced dosage for the genes encoding its subunits have defects in neural-tube closure similar to those in human spina bifida 3 , one of the most serious congenital birth defects. In contrast, BAF53b and the nBAF complex are essential for an evolutionarily conserved program of post-mitotic neural development and dendritic morphogenesis 4,5 . Here we show that this essential transition is mediated by repression of BAF53a by miR-9* and miR-124. We find that BAF53a repression is mediated by sequences in the 3′ untranslated region corresponding to the recognition sites for miR-9* and miR-124, which are selectively expressed in post-mitotic neurons. Mutation of these sites led to persistent expression of BAF53a and defective activity-dependent dendritic outgrowth in neurons. In addition, overexpression of miR-9* and miR-124 in neural progenitors caused reduced proliferation. Previous studies have indicated that miR-9* and miR-124 are repressed by the repressor-element-1-silencing transcription factor (REST, also known as NRSF) 6 . Indeed, expression of REST in post-mitotic neurons led to derepression of BAF53a, indicating that RESTmediated repression of microRNAs directs the essential switch of chromatin regulatory complexes.The ATP-dependent chromatin-remodelling complexes, typified by the yeast Swi/Snf complex, regulate chromatin assembly and accessibility 7,8 . The mammalian genome encodes nearly 30 different Swi2/Snf2-like ATPases, two of which, BRG1 and BRM are alternative subunits in complexes of 11 subunits termed BAF or mammalian SWI/SNF (mSWI/SNF) 3,4,9-11 . To understand the essential switch in subunit composition of these Correspondence and requests for materials should be addressed to G.R.C. (crabtree@stanford.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. complexes during neural development, we examined 180 kilobases (kb) around the BAF53a gene for transcriptional regulatory regions by replacing the first exon of BAF53a at the start codon with a destabilized nuclear enhanced green fluorescent protein (d2nucEGFP) in a BAF53a-containing bacterial artificial chromosome (BAC). The d2nucEGFP is followed by the stop codon and a 3′ untranslated region (UTR) so that EGFP expression refle...
We demonstrate editing of post-mitotic neurons in the adult mouse brain following injection of Cas9 ribonucleoprotein (RNP) complexes in the hippocampus, striatum and cortex. Engineered variants of Cas9 with multiple SV40 nuclear localization sequences enabled a tenfold increase in the efficiency of neuronal editing in vivo. These advances indicate the potential of genome editing in the brain to correct or inactivate the underlying genetic causes of neurological diseases.
The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against bacteriophage and function as powerful tools for genome editing in wide-ranging cell types. Here we present a third and fundamentally distinct RNA-guided platform, CRISPR-CasX, which uses a unique structure and mechanism for programmable double-stranded DNA cleavage. Biochemical and in vivo data demonstrate that CasX is active for E. coli and human genome modification. Eight cryo-EM structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and an unanticipated domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.
The CRISPR-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disrupting the enzyme’s binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the Estrogen Receptor α Ligand Binding Domain. This protein displayed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.
Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.CRISPR-Cas9 | genome engineering | split enzyme B acteria use RNA-guided adaptive immune systems encoded by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) genomic loci to defend against invasive DNA (1, 2). In type II CRISPR-Cas systems, a single enzyme called Cas9 is responsible for targeting and cleavage of foreign DNA (3). The ability to program Cas9 for DNA cleavage at sites defined by engineered single-guide RNAs (sgRNAs) (4) has led to its adoption as a robust and versatile platform for genome engineering (for recent reviews, see refs. 5-7).Cas9 contains two nuclease active sites that function together to generate DNA double-strand breaks (DSBs) at sites complementary to the 20-nt guide RNA sequence and adjacent to a protospacer adjacent motif (PAM). Structural studies of the Streptococcus pyogenes Cas9 showed that the protein exhibits a bilobed architecture comprising the catalytic nuclease lobe and the α-helical lobe of the enzyme (8). Electron microscopy (EM) studies and comparisons with X-ray crystal structures with and without a bound guide RNA and target DNA revealed a largescale conformational rearrangement of the two lobes relative to each other upon nucleic acid binding (8, 9). Strikingly, RNA binding induces the nuclease lobe to rotate ∼100°relative to the α-helical lobe, generating a nucleic-acid binding cleft that can accommodate DNA, and interactions between the two lobes seem to be mediated primarily through contacts with the bound nucleic acid rather than direct protein-protein contacts (8, 9). These observations suggested that the two structural lobes of Cas9 might be separable into independent polypeptides that retain the ability to assemble into an active enzyme complex. Such a system would enable analysis of the functionally distinct properties of each Cas9 structural region and might offer a unique mechanism for control...
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