Showing papers on "Genome editing published in 2015"

In vivo genome editing using Staphylococcus aureus Cas9

Abstract: The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

In vivo genome editing using Staphylococcus aureus Cas9

Abstract: The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining

Abstract: Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator-like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.

Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells

Abstract: The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.

Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

Abstract: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

Therapeutic genome editing: prospects and challenges

Abstract: Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

Abstract: Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins

Abstract: Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.

Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

Abstract: CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells

Abstract: Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.

Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids

Abstract: Genome editing applied to human intestinal organoids enables the study of the functional effects of mutations recurrent in human tumors.

Therapeutic genome editing: prospects and challenges

Abstract: Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.

In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9

Abstract: Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9)1 can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain.

BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

Abstract: Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

A Split Cas9 Architecture for Inducible Genome Editing and Transcription Modulation

Abstract: The RNA-guided endonuclease Cas9 has been harnessed as a tool for genome editing in mammalian cells1, 2. In addition, strategies employing catalytic inactive Cas9 can direct effector proteins to genomic targets3-5 to achieve transcriptional modulation. Here, we demonstrate that Cas9 can be split into two fragments and rendered chemically inducible by rapamycin sensitive dimerization domains for controlled reassembly to mediate genome editing and transcription modulation.

Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System

Abstract: 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 Acidaminococcus 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.

Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains

Abstract: CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases Current screening strategies target CRISPR-Cas9-induced mutations to the 5' exons of candidate genes, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies Here we overcome this limitation by targeting CRISPR-Cas9 mutagenesis to exons encoding functional protein domains This generates a higher proportion of null mutations and substantially increases the potency of negative selection We also show that the magnitude of negative selection can be used to infer the functional importance of individual protein domains of interest A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identifies six known drug targets and 19 additional dependencies A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting

Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

Abstract: Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future.

Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition

Abstract: CRISPR-Cas9 nucleases target specific DNA sequences using a guide RNA but also require recognition of a protospacer adjacent motif (PAM) by the Cas9 protein. Although longer PAMs can potentially improve the specificity of genome editing, they limit the range of sequences that Cas9 orthologs can target. One potential strategy to relieve this restriction is to relax the PAM recognition specificity of Cas9. Here we used molecular evolution to modify the NNGRRT PAM of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, showed robust genome editing activities at endogenous human target sites with NNNRRT PAMs, thereby increasing SaCas9 targeting range by two- to fourfold. Using GUIDE-seq, we show that wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. Our strategy for evolving PAM specificity does not require structural information and therefore should be applicable to a wide range of Cas9 orthologs.

High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes.

Abstract: Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro–assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.

Multiplex Genome-Edited T-cell Manufacturing Platform for “Off-the-Shelf” Adoptive T-cell Immunotherapies

Abstract: Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator-like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their αβ T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19(+) tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an "off-the-shelf" manner akin to other biologic immunopharmaceuticals

Photoactivatable CRISPR-Cas9 for optogenetic genome editing

Abstract: We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications.

Self‐Assembled DNA Nanoclews for the Efficient Delivery of CRISPR–Cas9 for Genome Editing

Abstract: CRISPR-Cas9 represents a promising platform for genome editing, yet means for its safe and efficient delivery remain to be fully realized. A novel vehicle that simultaneously delivers the Cas9 protein and single guide RNA (sgRNA) is based on DNA nanoclews, yarn-like DNA nanoparticles that are synthesized by rolling circle amplification. The biologically inspired vehicles were efficiently loaded with Cas9/sgRNA complexes and delivered the complexes to the nuclei of human cells, thus enabling targeted gene disruption while maintaining cell viability. Editing was most efficient when the DNA nanoclew sequence and the sgRNA guide sequence were partially complementary, offering a design rule for enhancing delivery. Overall, this strategy provides a versatile method that could be adapted for delivering other DNA-binding proteins or functional nucleic acids.

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation

Abstract: The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.

High-frequency, precise modification of the tomato genome

Abstract: The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA. Here, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences. High-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA.

Inducible in vivo genome editing with CRISPR-Cas9

Abstract: CRISPR-Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR-Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9(D10A) (Cas9n) variant can regulate the frequency and size of target gene modifications, respectively. Further, we show that this inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and, thus, provides a flexible and fast platform to study loss-of-function phenotypes in vivo.

High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system.

Abstract: Actinobacteria, particularly those of genus Streptomyces, remain invaluable hosts for the discovery and engineering of natural products and their cognate biosynthetic pathways. However, genetic manipulation of these bacteria is often labor and time intensive. Here, we present an engineered CRISPR/Cas system for rapid multiplex genome editing of Streptomyces strains, demonstrating targeted chromosomal deletions in three different Streptomyces species and of various sizes (ranging from 20 bp to 30 kb) with efficiency ranging from 70 to 100%. The designed pCRISPomyces plasmids are amenable to assembly of spacers and editing templates via Golden Gate assembly and isothermal assembly (or traditional digestion/ligation), respectively, allowing rapid plasmid construction to target any genomic locus of interest. As such, the pCRISPomyces system represents a powerful new tool for genome editing in Streptomyces.