Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate

The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 5′ ends of double-stranded DNA are recessed by λ exonuclease, leaving behind 3′ overhangs. Here, we propose an alternative in which lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via beta recombinase-catalyzed annealing at the replication fork. We support this by showing that single-stranded gene insertion cassettes are recombinogenic and that these cassettes preferentially target the lagging strand during DNA replication. Furthermore, a double-stranded DNA cassette containing multiple internal mismatches shows strand-specific mutations cosegregating roughly 80% of the time. These observations are more consistent with our model than with previously proposed models. Finally, by using phosphorothioate linkages to protect the lagging-targeting strand of a double-stranded DNA cassette, we illustrate how our new mechanistic knowledge can be used to enhance lambda Red recombination frequency. The mechanistic insights revealed by this work may facilitate further improvements to the versatility of lambda Red recombination.OVER the past decade, lambda Red recombination (“recombineering”) has been used as a powerful technique for making precisely defined insertions, deletions, and point mutations in Escherichia coli, requiring as few as 35 bp of homology on each side of the desired alteration (Thomason et al. 2007a; Sharan et al. 2009). With this system, single-stranded DNA (ssDNA) oligonucleotides have been used to efficiently modify E. coli chromosomal targets (Ellis et al. 2001; Costantino and Court 2003), BACs (Swaminathan et al. 2001), and plasmids (Thomason et al. 2007b), as well as to rapidly optimize a metabolic pathway coding for the production of lycopene (Wang et al. 2009). Furthermore, linear double-stranded DNA (dsDNA) recombineering has been used to replace chromosomal genes (Murphy 1998; Murphy et al. 2000), to disrupt gene function (Datsenko and Wanner 2000), and to develop novel cloning methods (Lee et al. 2001; Li and Elledge 2005). Large-scale dsDNA recombineering projects include creating a library of single-gene knockout E. coli strains (Baba et al. 2006) and removing 15% of the genomic material from a single E. coli strain (Posfai et al. 2006). Linear dsDNA recombineering has also been used to insert heterologous genes and entire pathways into the E. coli chromosome (Zhang et al. 1998; Wang and Pfeifer 2008) and BACs (Lee et al. 2001; Warming et al. 2005), including those used for downstream applications in eukaryotes (Chaveroche et al. 2000; Bouvier and Cheng 2009). However, despite the broad use of this method, the mechanism of lambda Red recombination has not achieved scientific consensus, particularly in the case of dsDNA recombination. A clearer understanding of the mechanism underlying this process could suggest ways to improve the functionality, ease, and versatility of lambda Red recombination.Three phage-derived lambda Red proteins are necessary for carrying out dsDNA recombination: Gam, Exo, and Beta. Gam prevents the degradation of linear dsDNA by the E. coli RecBCD and SbcCD nucleases; lambda exonuclease (Exo) degrades dsDNA in a 5′ to 3′ manner, leaving single-stranded DNA in the recessed regions; and Beta binds to the single-stranded regions produced by Exo and facilitates recombination by promoting annealing to the homologous genomic target site (Sawitzke et al. 2007). Current mechanisms claim that Exo binds to both 5′ ends of the dsDNA and degrades in both directions simultaneously to produce a double-stranded region flanked on both sides by 3′ overhangs (Sharan et al. 2009; Szczepanska 2009). However, a comprehensive explanation of how this construct ultimately recombines with the chromosome has not yet been advanced.Initially, it was proposed that this recombination occurs via strand invasion (Thaler et al. 1987). However, it has more recently been shown that strand invasion is unlikely to be the dominant mechanism in the absence of long regions of homology, as recombination remains highly proficient in a recA^- background (Yu et al. 2000). Furthermore, a detailed analysis of lambda Red recombination products showed characteristics consistent with strand annealing rather than a strand invasion model (Stahl et al. 1997). Finally, lambda Red dsDNA recombination has been shown to preferentially target the lagging strand during DNA replication, which suggests strand annealing rather than strand invasion (Lim et al. 2008; Poteete 2008).To explain these results, Court et al. (2002) proposed a strand-annealing model for insertional dsDNA recombination (Figure 1A), in which one single-stranded 3′ end anneals to its homologous target at the replication fork. The replication fork then stalls, due to the presence of a large dsDNA nonhomology (i.e., the insertion cassette). The stalled replication fork is ultimately rescued by the other replication fork traveling in the opposite direction around the circular bacterial chromosome. The other 3′ end of the recombinogenic DNA anneals to the homology region exposed by the second replication fork, forming a crossover structure, which is then resolved by unspecified E. coli enzymes (Court et al. 2002).Open in a separate windowFigure 1.—Previously proposed lambda Red-mediated dsDNA recombination mechanisms. Heterologous dsDNA is shown in green; Exo is an orange oval, and Beta is a yellow oval. In both mechanisms the recombination intermediate is proposed to be a dsDNA core flanked on either side by 3′ ssDNA overhangs. (A) The Court mechanism posits that (1) Beta facilitates annealing of one 3′ overhang to the lagging strand of the replication fork. (2) This replication fork then stalls and backtracks so that the leading strand can template switch onto the synthetic dsDNA. The heterologous dsDNA blocks further replication from this fork. (3) Once the second replication fork reaches the stalled fork, the other 3′ end of the integration cassette is annealed to the lagging strand in the same manner as prior. Finally, the crossover junctions must be resolved by unspecified E. coli enzymes (Court et al. 2002). (B) The Poteete mechanism suggests that (1) Beta facilitates 3′ overhang annealing to the lagging strand of the replication fork and (2) positions the invading strand to serve as the new template for leading-strand synthesis. This structure is resolved by an unspecified host endonuclease (red triangle), and (3) the synthetic dsDNA becomes template for both lagging and leading-strand synthesis. A second template switch must then occur at the other end of the synthetic dsDNA (Poteete 2008). The figure was adapted from the references cited.The Court mechanism was challenged by Poteete (2008), who showed that the dsDNA recombination of a linear lambda phage chromosome occurs readily onto a unidirectionally replicating plasmid, which does not have the second replication fork required by the Court mechanism (Court et al. 2002). Thus, Poteete proposed an alternate mechanism (Poteete 2008), termed “replisome invasion” (Figure 1B), in which a 3′ overhang of the Exo-processed dsDNA first anneals to its complementary sequence on the lagging strand of the recombination target. Subsequently, this overhang displaces the leading strand, thereby serving as the new template for leading-strand synthesis. The resulting structure is resolved by an unspecified endonuclease, after which the recombinogenic DNA becomes the template for the synthesis of both new strands. In the context of recombineering using a linear dsDNA cassette, the author indicates that a second strand-switching event must occur at the other end of the incoming dsDNA.While Poteete''s mechanism addresses some of the weaknesses of the Court mechanism, it remains largely speculative. This mechanism does not identify the endonuclease responsible for resolving the structure after the first template switching event, nor does it explain how the recombinogenic DNA and replication machinery form a new replication fork. Additionally, this template-switching mechanism would have to operate two times in a well-controlled manner, which may not be consistent with the high-recombination frequencies often observed (Murphy et al. 2000) for lambda Red-mediated dsDNA insertion. Finally, little experimental evidence has been advanced to directly support this hypothesis.To address the deficiencies in these mechanisms, we propose that lambda Red dsDNA recombination proceeds via a ssDNA intermediate rather than a dsDNA core flanked by 3′ overhangs (Figure 2). In this mechanism, Exo binds to one of the two dsDNA strands and degrades that strand completely, leaving behind full-length ssDNA. This ssDNA then anneals to its homology target at the lagging strand of the replication fork and is incorporated as part of the newly synthesized strand as if it were an Okazaki fragment. This process is analogous to the accepted mechanism for the lambda Red-mediated recombination of ssDNA oligonucleotides (Court et al. 2002) and, therefore, unifies the mechanisms for ssDNA and dsDNA recombination. Notably, our mechanism uses one replication fork for the incorporation of a full-length heterologous cassette, thereby addressing Poteete''s criticism of the Court mechanism.Open in a separate windowFigure 2.—Lambda Red mediated dsDNA recombination proceeds via a ssDNA intermediate. Instead of a recombination intermediate involving dsDNA flanked by 3′-ssDNA overhangs, we propose that one strand of linear dsDNA is entirely degraded by Exo (orange oval). Beta (yellow oval) then facilitates annealing to the lagging strand of the replication fork in place of an Okazaki fragment. The heterologous region does not anneal to the genomic sequence. This mechanism could account for gene replacement (as shown) or for insertions in which no genomic DNA is removed.The degradation of an entire strand by lambda Exo is feasible, given the highly processive nature of the enzyme (Subramanian et al. 2003). Whereas previously proposed mechanisms assume that both dsDNA ends are degraded approximately simultaneously, our hypothesis implies that some dsDNA molecules will be entirely degraded to ssDNA before a second Exo can bind to the other end. In this article, we demonstrate that single-stranded DNA is a viable recombinogenic intermediate with lagging-strand bias. Furthermore, we show that genetic information from one strand of a recombinogenic dsDNA cassette cosegregates during lambda Red-mediated recombination. These results provide strong support of our proposed mechanism.     

Photoactivatable CRISPR/Cas9 System

Russian Journal of Bioorganic Chemistry - A photoactivatable CRISPR/Cas9 system consisting of the Cas9 protein, synthetic 102-nt sgRNA or a pair of guide crRNA/tracrRNA, and blocking photocleavable...     

Efficient Gene Knockout in Goats Using CRISPR/Cas9 System

The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications.     

Targeted Chromosomal Translocations and Essential Gene Knockout Using CRISPR/Cas9 Technology in Caenorhabditis elegans

Many genes play essential roles in development and fertility; their disruption leads to growth arrest or sterility. Genetic balancers have been widely used to study essential genes in many organisms. However, it is technically challenging and laborious to generate and maintain the loss-of-function mutations of essential genes. The CRISPR/Cas9 technology has been successfully applied for gene editing and chromosome engineering. Here, we have developed a method to induce chromosomal translocations and produce genetic balancers using the CRISPR/Cas9 technology and have applied this approach to edit essential genes in Caenorhabditis elegans. The co-injection of dual small guide RNA targeting genes on different chromosomes resulted in reciprocal translocation between nonhomologous chromosomes. These animals with chromosomal translocations were subsequently crossed with animals that contain normal sets of chromosomes. The F1 progeny were subjected to a second round of Cas9-mediated gene editing. Through this method, we successfully produced nematode strains with specified chromosomal translocations and generated a number of loss-of-function alleles of two essential genes (csr-1 and mes-6). Therefore, our method provides an easy and efficient approach to generate and maintain loss-of-function alleles of essential genes with detailed genetic background information.     

Efficient Gene Targeting in Golden Syrian Hamsters by the CRISPR/Cas9 System

The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)—three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C—and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.     

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen.     

Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System

Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.     

The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.     

CRISPR/Cas9:基因编辑的新时代

基因编辑技术是通过核酸内切酶对基因组DNA进行定向改造的技术,可以实现对特定DNA碱基的缺失、替换等,常用的四种基因编辑工具分别是:巨型核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶以及CRISPR/Cas9系统.其中CRISPR/Cas9系统作为一种新型的基因组编辑技术具有组成简单、特异性好、切割效率高的优点.该文对...     

Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9

The mammalian prion protein (PrP, encoded by Prnp) is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer''s disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9), have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs). It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program.     

利用CRISPR/Cas9技术对miRNA-125a进行基因编辑的实验研究

近年来, CRISPR/Cas9技术被迅速应用于生物学研究的各个领域。miRNA-125a已被证实与生殖密切相关,研究者在自然流产的患者中发现了异常的miRNA-125a。该研究利用CRISPR/Cas9技术在人绒毛膜滋养层细胞HTR8-S/Vneo中对miRNA-125a进行了定点编辑。对于定点敲除,我们首先在pX458表达载体的基础上额外引入一组启动子和sgRNA骨架序列用于片段敲除,此后分别设计用于单点敲除和片段敲除的sgRNA进行表达载体的构建,表达载体转染HTR8细胞后通过流式分选和T7EI酶切鉴定阳性单克隆;对于定点敲入,我们在体外构建供体载体,与CRISPR表达载体共转染HTR8细胞,通过流式分选和PCR鉴定阳性单克隆。DNA测序结果显示,敲除及敲入阳性单克隆细胞系在指定区域的碱基序列发生了改变; RT-qPCR结果显示,阳性单克隆细胞系中mi RNA-125a-5p和mi RNA-125a-3p的表达量较野生型也发生了相应变化;体外实验还显示, miRNA-125a有抑制HTR8-S/Vneo细胞系增殖及迁移的功能。研究证实, CRISPR/Cas9技术在细胞系中编辑mi RNAs是有效可行的,且对于miRNA-125a的定点敲除,片段敲除的效果要好于单点敲除。     

Recent Progress in CRISPR/Cas9 Technology

The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 system, a simple and efficient tool for genome editing, has experienced rapid progress in its technology and applicability in the past two years. Here, we review the recent advances in CRISPR/Cas9 technology and the ways that have been adopted to expand our capacity for precise genome manipulation. First, we introduce the mechanism of CRISPR/Cas9, including its biochemical and structural implications. Second, we highlight the latest improvements in the CRISPR/Cas9 system, especially Cas9 protein modifications for customization. Third, we review its current applications, in which the versatile CRISPR/Cas9 system was employed to edit the genome, epigenome, or RNA of various organisms. Although CRISPR/Cas9 allows convenient genome editing accompanied by many benefits, we should not ignore the significant ethical and biosafety concerns that it raises. Finally, we discuss the prospective applications and challenges of several promising techniques adapted from CRISPR/Cas9.     

斑马鱼心脏发育候选基因Titin-Cap的CRISPR/Cas9打靶有效性分析

CRISPR/Cas9基因打靶技术是近几年发展起来的一种高效率的定向打靶技术,被认为是遗传领域的革命性技术。Titin-Cap基因是本实验室已初步鉴定的斑马鱼心脏发育候选基因,且国内外目前尚无斑马鱼Titin-Cap基因的敲除品系。为了研究Titin-Cap基因在心脏发育过程中的作用机制,我们利用CRISPR/Cas9基因打靶技术建立斑马鱼Titin-Cap基因的敲除品系。测序结果显示,注射了CRISPR/Cas9 gRNA的胚胎出现双峰,说明在打靶位点附近出现了碱基缺失或插入,证明我们设计的gRNA是有效的。对F0代突变体成鱼的筛选中,测序结果同样显示有阳性结果。这些结果说明用CRISPR/Cas9基因打靶技术成功敲除了斑马鱼Titin-Cap基因,获得了Titin-Cap基因敲除的嵌合体斑马鱼。     

建立CRISPR/Cas9慢病毒系统高效敲除人源AIP1基因

旨在构建AIP1基因CRISPR/Cas9敲除体系,获得高效、稳定、永久性AIP1敲除的人胚肾细胞株(293T)。针对AIP1的外显子设计3个20 bp的sg RNA(sp1、sp2和sp3)。与PX458载体连接,构建PX458-sg RNA敲除表达载体。T7E1实验评估敲除效率。将敲除效率最高的sg RNA与lenti CRISPRv2慢病毒载体连接,构建lenti CRISPRv2-sg RNA敲除表达载体,将阳性重组质粒导入病毒包装细胞293T中,收集病毒上清液转染293T细胞。对敲除成功的293T细胞通过有限稀释法构建敲除AIP1基因的稳定细胞株。Western blot测定转染后293T细胞中AIP1蛋白的表达量。结果显示,测序证实AIP1的3个靶序列分别正确插入PX458表达载体中,T7E1实验证实AIP1sg RNAsp2靶向敲除效率最高;成功构建lenti CRISPRv2-sg RNAsp2 AIP1敲除表达载体,并包装病毒,感染293T细胞;Western blot证实获得稳定的AIP1基因表达缺失的293T细胞株。建立了能稳定敲除AIP1基因的CRISPR/Cas9慢病毒系统,成功获得AIP1敲除的293T稳定细胞株。     

基于CRISPR/Cas9系统对斑马鱼hoxb4基因的编辑及其突变体基因型的初筛

采用高效基因编辑系统CRISPR/Cas9构建hoxb4基因敲除斑马鱼模型,进行hoxb4基因功能的研究。根据hoxb4基因的一号外显子的正义链及反义链设计3个长20 bp的sg RNA,分别靶向ExonⅠ的192#位点,244#位点及313#位点。化学合成sg RNA的寡核苷酸序列,经过酶切克隆进p T7-g RNA质粒中,构建g RNA的体外转录载体并通过体外转录得到靶位点的g RNA。将质粒p SP6-2s NLS-sp Cas9线性化然后在体外转录得到Cas9的m RNA并进行加A尾,将以上靶位点的g RNA与Cas9的m RNA共注射入单细胞期的斑马鱼胚胎内,提取基因组DNA,PCR扩增出目的基因片段并使用T7EI酶切测效,最后将PCR产物连入p MD19-T simple载体中,挑取阳性克隆进行菌落PC R鉴定,然后经Sanger测序检测突变类型。结果显示,靶位点的sg RN A寡核苷酸双链成功连入p T7-g RNA质粒中且序列正确;其中靶向ExonⅠ的313#位点的sg RNA可成功编辑斑马鱼hoxb4基因,T7 EⅠ检测其敲除效率高达26.5%,并测序得到4种阳性突变型。通过CRISPR/Cas9系统成功编辑斑马鱼hoxb4基因并测序鉴定其突变类型,为HOXb4基因功能的研究提供了可靠的基因敲除方法。     

Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System

CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9 system, which is a newly developed technology for targeted genome modification, has been successfully used in a number of species. In this study, we applied this technology to carry out targeted genome modification in maize. A marker gene Zmzb7 was chosen for targeting. The sg RNA-Cas9 construct was transformed into maize protoplasts, and indel(insertion and deletion) mutations could be detected. A mutant seedling with an expected albino phenotype was obtained from screening 120 seedlings generated from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated. Twelve sites with different expression levels in maize centromeres or pericentromere regions were selected. The sg RNACas9 constructs were transformed into protoplasts followed by sequencing the transformed protoplast genomic DNA. The results show that the genes in heterochromatic regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no PAM(protospacer adjacent motif) or having more than two mismatches. Together, our results show that the CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic and heterochromatic regions in maize.     

基于CRISPR/Cas9系统对干酪乳杆菌进行红色荧光蛋白标记

本实验利用CRISPR/Cas9系统对干酪乳杆菌(Lactobacillus casei) LC2W进行红色荧光蛋白(red fluorescent protein,RFP)标记,用于研究干酪乳杆菌在肠道内的分布和定植状况,评价其作为益生菌的功能。首先,基于本实验室已有的干酪乳杆菌CRISPR/Cas9编辑质粒pLCNICK-1628构建重组质粒pLCNICK-1628-RFP,电转入干酪乳杆菌LC2W感受态细胞中,使干酪乳杆菌基因组中的LC2W-1628基因被红色荧光蛋白基因替换,从而使干酪乳杆菌LC2W能表达出红色荧光蛋白。得到红色荧光标记的干酪乳杆菌LC2W突变株后,测定了其荧光强度-OD600标准曲线,发现RFP在干酪乳杆菌LC2W中能稳定表达。