Multiplex gene precise editing and large DNA fragment deletion by the CRISPR‐Cas9‐TRAMA system in edible mushroom Cordyceps militaris

The medicinal mushroom Cordyceps militaris contains abundant valuable bioactive ingredients that have attracted a great deal of attention in the pharmaceutical and cosmetic industries. However, the development of this valuable mushroom faces the obstacle of lacking powerful genomic engineering tools. Here, by excavating the endogenous tRNA‐processed element, introducing the extrachromosomal plasmid and alongside with homologous template, we develop a marker‐free CRISPR‐Cas9‐TRAMA genomic editing system to achieve the multiplex gene precise editing and large synthetic cluster deletion in C. militaris. We further operated editing in the synthetases of cordycepin and ergothioneine to demonstrate the application of Cas9‐TRAMA system in protein modification, promoter strength evaluation and 10 kb metabolic synthetic cluster deletion. The Cas9‐TRAMA system provides a scalable method for excavating the valuable metabolic resource of medicinal mushrooms and constructing a mystical cellular pathway to elucidate the complex cell behaviours of the edible mushroom.

This study identified the endogenic tRNA processed element of mushroom crop Cordyceps militaris, and proved the in vivo generation of gRNA by promoter PtrpC could highly improve the editing efficiency of genomic editing. By introducing the extra‐chromosomal plasmid and alongside with homologous template, a marker‐free CRISPR‐Cas9‐TRAMA genomic editing system was built to achieve the multiplex gene precise editing and large synthetic cluster deletion in C. militaris.     

CRISPR-Cas9驱动的基因编辑新纪元

在自然界生物长期的进化过程中,细菌和古细菌演化出了一种适应性免疫系统用以抵御外源病毒与质粒的入侵,该系统由成簇规律间隔的短回文重复序列与相关基因组成,称之为CRISPR-Cas。近年来,这一领域突飞猛进,如今已经发展成为一种功能强大的基因编辑工具并在生物学及其相关领域得到广泛应用。本文重点综述了近年来CRISPR-Cas9系统在基因编辑、基因调节以及作为体外工具酶和特异性等方面的若干前沿进展。     

Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways

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Multiplex CRISPR-Cas9 editing of DNA methyltransferases in rice uncovers a class of non-CG methylation specific for GC-rich regions

DNA methylation in the non-CG context is widespread in the plant kingdom and abundant in mammalian tissues such as the brain and pluripotent cells. Non-CG methylation in Arabidopsis thaliana is coordinately regulated by DOMAINS REARRANGED METHYLTRANSFERASE (DRM) and CHROMOMETHYLASE (CMT) proteins but has yet to be systematically studied in major crops due to difficulties in obtaining genetic materials. Here, utilizing the highly efficient multiplex CRISPR-Cas9 genome-editing system, we created single- and multiple-knockout mutants for all the nine DNA methyltransferases in rice (Oryza sativa) and profiled their whole-genome methylation status at single-nucleotide resolution. Surprisingly, the simultaneous loss of DRM2, CHROMOMETHYLASE3 (CMT2), and CMT3 functions, which completely erases all non-CG methylation in Arabidopsis, only partially reduced it in rice. The regions that remained heavily methylated in non-CG contexts in the rice Os-dcc (Osdrm2/cmt2/cmt3a) triple mutant had high GC contents. Furthermore, the residual non-CG methylation in the Os-dcc mutant was eliminated in the Os-ddccc (Osdrm2/drm3/cmt2/cmt3a/cmt3b) quintuple mutant but retained in the Os-ddcc (Osdrm2/drm3/cmt2/cmt3a) quadruple mutant, demonstrating that OsCMT3b maintains non-CG methylation in the absence of other major methyltransferases. Our results showed that OsCMT3b is subfunctionalized to accommodate a distinct cluster of non-CG-methylated sites at highly GC-rich regions in the rice genome.

Examination of knockout mutants reveals that rice methyltransferases have subfunctionalized to accommodate a distinct cluster of non-CG-methylated sites at highly GC-rich regions in the rice genome.     

CRISPR/Cas9系统在基因组DNA片段编辑中的应用

源于细菌和古菌的Ⅱ型成簇规律间隔短回文重复系统[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9),CRISPR/Cas9]近年被改造成为基因组定点编辑的新技术。由于它具有设计简单、操作方便、费用低廉等巨大优势,给遗传操作领域带来了一场革命性的改变。本文重点介绍了CRISPR/Cas9系统在基因组DNA片段靶向编辑方面的研究和应用,主要包括DNA片段的删除、反转、重复、插入和易位,这一有效的DNA片段编辑方法为研究基因功能、调控元件、组织发育和疾病发生发展提供了有力手段。本文最后展望了Ⅱ型CRISPR/Cas9系统的应用前景和其他类型CRISPR系统的应用潜力,为开展利用基因组DNA片段靶向编辑进行基因调控和功能研究提供参考。     

Multiplex gene editing of the <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> genome using the CRISPR-Cas9 system

Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.     

CRISPR-Cas9基因编辑技术在病毒感染疾病治疗中的应用

CRISPR-Cas9基因编辑技术是基于细菌或古细菌CRISPR介导的获得性免疫系统衍生而来,由一段RNA通过碱基互补配对识别DNA,指导Cas9核酸酶切割识别的双链DNA,诱发同源重组或非同源末端链接,进而实现在目的DNA上进行编辑。病毒通过特异的受体侵染细胞,其基因组在细胞内发生复制、转录、翻译等过程完成其生活周期,某些DNA病毒或逆转录病毒基因组会整合到宿主基因组中。基因治疗是病毒感染疾病治疗的新趋势。因此,基因编辑技术在持续感染的病毒或潜伏感染病毒疾病治疗中具有重大的潜在意义。文章主要从CRISPR-Cas9作用机制以及在病毒感染疾病治疗中的应用等方面进行了综述。     

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc^−/−) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.     

Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens

Plant Molecular Biology - We mutated all seven Physcomitrium (Physcomitrella) patens phytochrome genes using highly-efficient CRISPR-Cas9 procedures. We thereby identified phy5a as the phytochrome...     

Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFP^KI) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFP^KI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.     

Seamless deletion of a large DNA fragment in the taxol-producing fungus Pestalotiopsis microspora

To reduce the unnecessary gene clusters in the taxol-producing fungus Pestalotiopsis microspora, we report the development of an effective DNA deletion method that relies on a deletion cassette constructed with the Gateway-technique and overlap extension PCR, using the orotidine 5′-phosphate decarboxylase as recyclable marker for selection. By this approach, two adjacent DNA sequences can be sequentially deleted in a single transformation mediated by Agrobacterium tumefaciens, resulting in the deletion of a large DNA fragment. Additionally, the selection marker is spontaneously eliminated in this process. We used this method to successfully remove the mus53 locus of P. microspora.     

A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated nuclease 9(Cas9)-based genome editing tool pCas/pTargetF system that we establi...     

Heat and light stresses affect metabolite production in the fruit body of the medicinal mushroom Cordyceps militaris

Applied Microbiology and Biotechnology - Cordyceps militaris is a highly valued edible and medicinal fungus due to its production of various metabolites, including adenosine, cordycepin,...     

Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in Lesch-Nyhan families

The Lesch-Nyhan (LN) syndrome is a genetically lethal human neurological disease that results from mutations that inactivate the hypoxanthine phosphoribosyltransferase (HPRT) gene. The elucidation of the complete DNA sequence of the human HPRT gene locus has enabled the construction of multiple oligonucleotide primer sets for the simultaneous in vitro amplification of all nine HPRT exons. The multiplex polymerase chain reaction provides a facile assay for the detection of HPRT exon deletions and the reaction products can be analyzed by direct automated fluorescent DNA sequencing to identify subtle alterations in the gene. Alterations have been identified in the HPRT genes from 15 independent LN cases, and 10 LN family studies were performed. The sequencing method uses solid supports and is sufficiently simple and sensitive to be a favored approach for LN diagnosis. LN heterozygotes can be diagnosed without reference to the affected male. In addition, these procedures will be useful for somatic mutagenesis studies.     

Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene

The CRISPR-Cas9 system uses guide RNAs to direct the Cas9 endonuclease to cleave target sequences. It can, in theory, target essentially any sequence in a genome, but the efficiency of the predicted guide RNAs varies dramatically. If no targeted cells are obtained, it is also difficult to know why the experiment fails. We have developed a transient transfection based method to enrich successfully targeted cells by co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene. Cells are transfected with two guide RNAs that target respectively HPRT and the gene of interest. HPRT targeted cells are selected by resistance to 6-thioguanine (6-TG) and then examined for potential alterations to the gene targeted by the co-transfected guide RNA. Alterations of many genes, such as AAVS1, Exo1 and Trex1, are highly enriched in the 6-TG resistant cells. This method works in both HCT116 cells and U2OS cells and can easily be scaled up to process multiple guide RNAs. When co-targeting fails, it is straightforward to determine whether the target gene is essential or the guide RNA is ineffective. HPRT co-targeting thus provides a simple, efficient and scalable way to enrich gene targeting events and to identify the cause of failure.The CRISPR-Cas9 system is a revolutionary technology for gene targeting in cells (1–3). It consists of two components: a guide RNA and the Cas9 endonuclease that respectively pairs with the target sequence and then cleaves it (4–6). The guide RNA contains 19 nt that in theory can be custom designed to target almost any sequence in a genome (5–8). In practice, the effectiveness of the predicted guide RNAs varies dramatically (8). This problem, when compounded by other commonly encountered problems such as poor efficiency of DNA transfection or low titer of viruses, can make the isolation of successfully targeted cells a really laborious task (9). Low transfection efficiency can be improved by special techniques like nucleofection (6), but they are expensive and not readily available to all labs. A common method to enrich successfully targeted cells is to use drug resistance or green fluorescent protein (GFP) markers to select for cells that have integrated the guide RNA-expressing DNA into chromosomes (7,10). Fluorescence-activated cell sorting (FACS) sorting only enriches cells expressing high levels of GFP but not necessarily Cas9, while integration of foreign DNA, especially viral DNA, into the chromosome is itself an alteration to the genome and might cause oncogenic transformation. In some cases, Cas9 is also integrated into the genome and constitutively expressed (7). Persistent expression of the guide RNA in combination with Cas9 might lead to increasing probabilities of off-target cleavages over long-term culturing (11). In addition, if no targeted cells are obtained, it is difficult to track down the cause of failure. The guide RNA might be ineffective or the correctly targeted cells might have died off and only cells not expressing the guide RNA might have survived.To overcome these shortcomings, we have developed a transient plasmid DNA transfection-based method to enrich successfully targeted cells without the need for DNA integration into chromosomes by co-targeting the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene. This gene encodes a protein that catalyzes the conversion of hypoxanthine to inosinemonophosphate and guanine to guanosine monophosphate in the non-essential purine salvage pathway (12). HPRT⁺ cells are sensitive to 6-thioguanine (6-TG), which can be converted to the nucleotide form by HPRT and incorporated into DNA by DNA polymerase, killing cells by a process involving postreplicative mismatch repair (13–15). The strategy is to transfect cells with two plasmids that express respectively a HPRT guide RNA and a guide RNA for the gene of interest. Cas9 can be expressed from the gene on a separate plasmid, a plasmid carrying the HPRT gRNA or integrated into the chromosome (if such a cell line is already available). If a cell becomes resistant to 6-TG, it would suggest that this cell should also be competent to target the gene of interest as long as the gRNA is effective. Thus if the targeted gene is not altered in the resulting 6-TG resistant cells, it would suggest that the guide RNA is ineffective. On the other hand, if no 6-TG resistant cells can be obtained by co-targeting, it would suggest that the gene of interest might be essential.We have tested this method with guide RNAs for HPRT and the non-essential AAVS1 locus in HCT116 cells, a colorectal cancer cell line with a near diploidic karyotype (16). The results showed a dramatic enrichment of AAVS1 targeting events from below detection without co-targeting to over 80% with co-targeting. Other non-essential genes such as Trex1 and Exo1 were also successfully enriched by HPRT co-targeting. The method also worked in U2OS cells, an osteosarcoma cell line with a complicated karyotype (17). Co-targeting with guide RNAs for DNA topoisomerase 2α (Top2α) gave rise to no 6-TG resistant cells, which is consistent with Top2α being essential for cell proliferation (18). On the other hand, co-targeting with some other guide RNAs gave rise to plenty of 6-TG resistant cells but no alteration to the intended sequences, suggesting that the guide RNAs were ineffective. Together, these results demonstrate that co-targeting the HPRT gene provides a simple and efficient method to enrich successfully targeted cells. It can also be easily used to evaluate the effectiveness of guide RNAs and the essentialness of target genes.