The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas Class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here we report characterization of Cpf1, a putative Class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes, from Acidominococcus and Lachnospiraceae, with efficient genome editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
The RNA-guided endonuclease Cas9 is a versatile genome editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome editing applications requiring a high level of specificity.
The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Cas effector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.
The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Cas effector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific † Correspondence should be addressed to zhang@broadinstitute.org (F.Z.) and koonin@ncbi.nlm.nih.gov (E.V.K.). * These authors contributed equally to this work. Almost all archaea and about half of bacteria possess Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas) adaptive immune systems (1, 2), which protect microbes from viruses and other invading DNA through three steps: (i) adaptation, i.e., insertion of foreign nucleic acid segments (spacers) into the CRISPR array in between pairs of direct repeats (DRs), (ii) transcription and processing of the CRISPR array to produce mature CRISPR RNAs (crRNAs), and (iii) interference, whereby Cas enzymes are guided by the crRNAs to target and cleave cognate sequences in the respective invader genomes (3-5). All CRISPR-Cas systems characterized to date follow these three steps, although the mechanistic implementation and proteins involved in these processes display extensive diversity. Supplementary Materials Supplementary Materials and MethodsThe CRISPR-Cas systems are broadly divided into two classes on the basis of the architecture of the interference module: Class 1 systems rely on multi-subunit protein complexes whereas Class 2 systems utilize single effector proteins (1). Within these two classes, types and subtypes are delineated according to the presence of distinct signature genes, protein sequence conservation, and organization of the respective genomic loci. (Fig. 1A). This resulted in a library of 3,473 spacer sequences (along with 490 non-targeting guides designed to have a Levenshtein distance of ≥8 with respect to the MS2 and E. coli genomes) which we inserted between pLshC2c2 direct repeats (DRs). After transformation in of this construct into E. coli, we infected cells with varying dilutions of MS2 (10 −1 , 10 −3 , and 10 −5 ) and analyzed surviving cells to determine the spacer sequences carried by cells that survived the infection. Cells carrying spacers that confer robust interference against MS2 are expected to proliferate faster than those that lack such sequences. Following growth for 16 hours, we identified a number of spacers that were consistently enriched across three independent infection replicas in both the 10 −1 and 10 −3 dilution conditions, suggesting that they enabled interference against MS2. Specifically, 147 and 150 spacers showed >1.25 log 2 -fold enrichment in all three replicates for the 10 −1 and 10 −3 phage dilutions, respectively; of these two groups of...
Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA, at 2.8 Å resolution. AsCpf1 adopts a bilobed architecture, with the RNA–DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences, thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for the cleavage of the non-target and target strands, respectively, and jointly generate staggered DNA double-strand breaks. AsCpf1 recognizes the 5′-TTTN-3′ protospacer adjacent motif by base and shape readout mechanisms. Our findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1, and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox.
SUMMARY CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two Class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 or Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.
The foundational adenine base editors (e.g. ABE7.10) enable programmable C•G to T•A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG PAM sites, ABE8s result in ~1.5x higher editing at protospacer positions A5-A7 and ~3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2, with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification which is maintained when multiplexed across three loci. Delivered as mRNA, ABE8s induce no significant levels of sgRNA-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.
SUMMARY Autism spectrum disorder (ASD) is a heterogeneous disease, but genetically defined models can provide an entry point to studying the molecular underpinnings of this disorder. We generated germline mutant mice with loss-of-function mutations in Chd8, a de novo mutation strongly associated with ASD, and demonstrate that these mice display hallmark ASD behaviors, macrocephaly, and craniofacial abnormalities similar to patient phenotypes. Chd8+/− mice display a broad, brain-region specific dysregulation of major regulatory and cellular processes, most notably histone and chromatin modification, mRNA and protein processing, Wnt signaling, and cell cycle regulation. We also find altered synaptic physiology in medium spiny neurons of the nucleus accumbens. Perturbation of Chd8 in adult mice recapitulates improved acquired motor learning behavior found in Chd8+/− animals, suggesting a role for CHD8 in adult striatal circuits. These results support a mechanism linking chromatin modification to striatal dysfunction and the molecular pathology of ASD.
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