A possible aid in targeted insertion of large DNA elements by CRISPR/Cas in mouse zygotes

The CRISPR/Cas system has rapidly emerged recently as a new tool for genome engineering, and is expected to allow for controlled manipulation of specific genomic elements in a variety of species. A number of recent studies have reported the use of CRISPR/Cas for gene disruption (knockout) or targeted insertion of foreign DNA elements (knock‐in). Despite the ease of simple gene knockout and small insertions or nucleotide substitutions in mouse zygotes by the CRISPR/Cas system, targeted insertion of large DNA elements remains an apparent challenge. Here the generation of knock‐in mice with successful targeted insertion of large donor DNA elements ranged from 3.0 to 7.1 kb at the ROSA26 locus using the CRISPR/Cas system was achieved. Multiple independent knock‐in founder mice were obtained by injection of hCas9 mRNA/sgRNA/donor vector mixtures into the cytoplasm of C57BL/6N zygotes when the injected zygotes were treated with an inhibitor of actin polymerization, cytochalasin. Successful germ line transmission of three of these knock‐in alleles was also confirmed. The results suggested that treatment of zygotes with actin polymerization inhibitors following microinjection could be a viable method to facilitate targeted insertion of large DNA elements by the CRISPR/Cas system, enabling targeted knock‐in readily attainable in zygotes. genesis 54:65–77, 2016. © 2016 Wiley Periodicals, Inc.     

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。     

Generation of a Double KO Mouse by Simultaneous Targeting of the Neighboring Genes Tmem176a and Tmem176b Using CRISPR/Cas9:Key Steps from Design to Genotyping

The CRISPR/Cas9 system has been tailored to a revolutionary genetic tool because of its remarkable simplicity and efficacy.While complex genome editing in the mouse since the 1990 s has been dominated by the use of embryonic stem(ES) cells,CRISPR/Cas9 now offers a versatile and fast approach to precisely modify virtually any DNA regions directly in mouse zygotes.Yet,this relative simplicity does not preclude a conscientious preparatory work that is often neglected when initiating a project.Here,we describe the key steps leading to successful generation of a double knockout(KO) mouse by simultaneously targeting two homolog genes,Tmem176 a and Tmem176 b,which are located in the same genomic locus.Additionally,we show that similar efficiency can be obtained in a mixed genetic background or directly in the C57BL/6 inbred strain.Thus,presented as a detailed case study that should be helpful to the non-specialists,we focus on the genotyping strategy to anticipate the various possibilities.     

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.     

Genome-Wide Study of Structural Variants in Bovine Holstein,Montbéliarde and Normande Dairy Breeds

High-throughput sequencing technologies have offered in recent years new opportunities to study genome variations. These studies have mostly focused on single nucleotide polymorphisms, small insertions or deletions and on copy number variants. Other structural variants, such as large insertions or deletions, tandem duplications, translocations, and inversions are less well-studied, despite that some have an important impact on phenotypes. In the present study, we performed a large-scale survey of structural variants in cattle. We report the identification of 6,426 putative structural variants in cattle extracted from whole-genome sequence data of 62 bulls representing the three major French dairy breeds. These genomic variants affect DNA segments greater than 50 base pairs and correspond to deletions, inversions and tandem duplications. Out of these, we identified a total of 547 deletions and 410 tandem duplications which could potentially code for CNVs. Experimental validation was carried out on 331 structural variants using a novel high-throughput genotyping method. Out of these, 255 structural variants (77%) generated good quality genotypes and 191 (75%) of them were validated. Gene content analyses in structural variant regions revealed 941 large deletions removing completely one or several genes, including 10 single-copy genes. In addition, some of the structural variants are located within quantitative trait loci for dairy traits. This study is a pan-genome assessment of genomic variations in cattle and may provide a new glimpse into the bovine genome architecture. Our results may also help to study the effects of structural variants on gene expression and consequently their effect on certain phenotypes of interest.     

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

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.     

Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.     

Off‐target assessment of CRISPR‐Cas9 guiding RNAs in human iPS and mouse ES cells

The CRISPR‐Cas9 system consists of a site‐specific, targetable DNA nuclease that holds great potential in gene editing and genome‐wide screening applications. To apply the CRISPR‐Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterized the rate of Cas9 off‐target activity in typical Cas9 experiments in two human and one mouse cell lines. We analyzed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ～90 predicted off‐target sites per gRNA. In a Cas9‐based knockout experiment, gRNAs induced detectable Cas9 cutting activity in all on‐target sites and in only a few off‐target sites genome‐wide in human 293FT, human‐induced pluripotent stem (hiPS) cells, and mouse embryonic stem (ES) cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on‐target and off‐target sites. In clonal Cas9 cutting analysis in mouse ES cells, biallelic Cas9 cutting was observed with low off‐target activity. Our results show that off‐target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off‐target site. Off‐target Cas9 activity can be minimized by selecting gRNAs with few off‐target sites of near complementarity. genesis 53:225–236, 2015. © 2014 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein

The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9–FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.     

A versatile reporter system for CRISPR-mediated chromosomal rearrangements

Although chromosomal deletions and inversions are important in cancer, conventional methods for detecting DNA rearrangements require laborious indirect assays. Here we develop fluorescent reporters to rapidly quantify CRISPR/Cas9-mediated deletions and inversions. We find that inversion depends on the non-homologous end-joining enzyme LIG4. We also engineer deletions and inversions for a 50 kb Pten genomic region in mouse liver. We discover diverse yet sequence-specific indels at the rearrangement fusion sites. Moreover, we detect Cas9 cleavage at the fourth nucleotide on the non-complementary strand, leading to staggered instead of blunt DNA breaks. These reporters allow mechanisms of chromosomal rearrangements to be investigated.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0680-7) contains supplementary material, which is available to authorized users.     

Plant genome editing: ever more precise and wide reaching

Genome-editing technologies consisting of targeted mutagenesis and gene targeting enable us to modify genes of interest rapidly and precisely. The discovery in 2012 of CRISPR/Cas9 systems and their development as sequence-specific nucleases has brought about a paradigm shift in biology. Initially, CRISPR/Cas9 was applied in targeted mutagenesis to knock out a target gene. Thereafter, advances in genome-editing technologies using CRISPR/Cas9 developed rapidly, with base editing systems for transition substitution using a combination of Cas9 nickase and either cytidine or adenosine deaminase being reported in 2016 and 2017, respectively, and later in 2021 bringing reports of transversion substitution using Cas9 nickase, cytidine deaminase and uracil DNA glycosylase. Moreover, technologies for gene targeting and prime editing systems using DNA or RNA as donors have also been developed in recent years. Besides these precise genome-editing strategies, reports of successful chromosome engineering using CRISPR/Cas9 have been published recently. The application of genome editing to crop breeding has advanced in parallel with the development of these technologies. Genome-editing enzymes can be introduced into plant cells, and there are now many examples of crop breeding using genome-editing technologies. At present, it is no exaggeration to say that we are now in a position to be able to modify a gene precisely and rearrange genomes and chromosomes in a predicted way. In this review, we introduce and discuss recent highlights in the field of precise gene editing, chromosome engineering and genome engineering technology in plants.     

利用CRISPR/Cas9对基因组中高度同源DNA片段编辑多样性的遗传学研究

在基因组中,编码区存在许多高度相似的基因簇或基因群(多拷贝基因),非编码区也存在大量的重复序列。这些重复序列能通过改变染色体的三维结构调控基因的转录,对于生物体的遗传与进化起到了重要的作用。其高度同源的特征使得利用CRISPR/Cas9技术进行基因组编辑时面临更加复杂的状况。如果编辑的片段是二倍体或多倍体,还会产生各条染色单体上的编辑情况不相同的现象。为此本文选择了2个位于同一染色体相距11 kb的高度同源300 bp片段(L1和L2)进行CRISPR介导的DNA片段编辑。采用一对sgRNA(分别共同靶向两片段的上、下游位点)引导Cas9对HepG2细胞两个高度相似的DNA片段进行切割。片段编辑的细胞进一步单克隆化后,对获得的22个L1/L2编辑的CRISPR单克隆细胞株进行详细的基因型鉴定。结果发现除了这两个DNA片段本身被删除外,它们之间的大片段也存在被删除的现象,三个片段的各种反转组合也很频繁。该研究结果对于采用CRISPR/Cas9系统编辑多拷贝基因或重复序列,尤其是对二倍体或多倍体生物进行基因组编辑时具有重要的借鉴和参考价值。     

Efficient multinucleotide deletions using deaminase-Cas9 fusions in human cells

CRISPR/Cas9 system is a robust genome editing platform in biotechnology and medicine. However, it generally produces small insertions/deletions (indels, typically 1–3 bp) but rarely induces larger deletions in specific target sites. Here, we report a cytidine deaminase-Cas9 fusion-induced deletion system (C-DEL) and an adenine deaminase-Cas9 fusion-induced deletion system (A-DEL) by combining Cas9 with rat APOBEC1 (rA1) and TadA 8e, respectively. Both C-DEL and A-DEL improve the efficiency of deletions compared with the conventional Cas9 system in human cells. In addition, the C-DEL system generates a considerable fraction of predictable multinucleotide deletions from 5′-deaminated C bases to the Cas9-cleavage site and increases the proportion of larger deletions at the target loci. Taken together, the C-DEL and A-DEL systems provide a practical strategy for producing efficient multinucleotide deletions, expanding the CRISPR/Cas9 toolsets for gene modifications in human cells.     

Is mouse embryonic stem cell technology obsolete?

Injection of recombinant Cas9 protein and synthetic guide RNAs into mouse zygotes has been shown to facilitate gene disruption and knock-ins using the CRISPR system. These technologies may soon displace genetic modification using embryonic stem cells.     

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.     

Hypomorphic phenotype of <Emphasis Type="Italic">Foxn1</Emphasis> gene-modified rats by CRISPR/Cas9 system

The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.