(Kries et al. 2014). Attempts to drastically change the substrate specificity have resulted in a decrease in the A domain activity (Chen et al. 2009). In vivo efforts to create NRP analogues with variations in AA composition by tar- geted mutagenesis of A domains appear to be even more challenging (Eppelmann et al. 2002; Uguru et al. 2004; Han et al. 2012; Thirlway et al. 2012). In this study, we attempted to rationally and systematically change the speci- ficity of the glycine- incorporating A domain in the NRPS module of the kalimantacin biosynthetic assembly line by using a new targetedmutagenesis approach and a specificity- conferring code designed for use in Pseudomonas. The functionality and specificity of the mutated A domains were subsequently analyzed by ultrahigh- resolution LC- MS analysis of culture supernatants.
Targetedmutagenesis by zinc-finger nucleases (ZFNs) can be used to generate knock-out mamma- lian cell lines with high efficiency. A number of different methods have been developed for the de- sign and assembly of gene-specific ZFNs, making them easily accessible to researchers. In this study, we used ZFNs assembled through the CoDA (context-dependent assembly) platforms to generate mutant caprine fetal fibroblasts cells for the BLG gene. ZFN plasmid was introduced into the caprine fetal fibroblasts cell by electroporation. ZFN-induced cleavage of the target sequence was conﬁrmed by the Surveyor nuclease assay analysis. Sequence analysis revealed that ZFN- induced mutations such as base insertion, deletion, or substitution were generated in the ZFN cleavage site of BLG. The simplicity and efficacy of CoDA will enable broad application of ZFN technology. This technique could be used with homologous arm, which may target foreign genes into the BLG locus at higher efficiency.
ABSTRACT Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate ef ﬁ cient targetedmutagenesis in medaka ( Oryzias latipes ), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 ( PARK7 ). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high ef ﬁ ciency. This induction of mutations occurred in a dose-dependent manner. All screened G0 ﬁ sh injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efﬁciency (44–100%). We also conﬁrmed that these TALENs induced site-speciﬁc muta- tions because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1 Δ7/Δ7 ﬁsh that carried a TALEN-induced frameshift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N terminus and 63 amino acids at the C terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efﬁciently establish knockout medaka strains. This is the ﬁrst report of targetedmutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics.
Genetic targets for improving FFA production in S. elongatus PCC 7942 include both general stress re- sponse genes as well as response mechanisms that may be specific for FFA-induced stress. Stress response genes that were highly conserved across the cyanobac- terial transcriptomic studies also had high fold change values. In particular, two genes were differentially regulated in 13 of the compared stress conditions: the high light in- ducible protein (Synpcc7942_1997, average +11.47 fold change) and a hypothetical protein (Synpcc7942_0551, average −4.52 fold change). Another 4 genes were up- regulated in 11 or 12 of the stress conditions: the heat shock protein Hsp20 (Synpcc7942_2401, average +102.59 fold change), the NAD(P)H-quinone oxidoreductase subunit 4 (Synpcc7942_1439, average +10.54 fold change), RNA poly- merase sigma factor SigD (Synpcc7942_0672, average +4.22 fold change), and Beta-Ig-H3/fasciclin (Synpcc7942_1606, average +12.44 fold change). These 6 genes are candidate targets for boosting the native stress response mechanisms of S. elongatus PCC 7942. The hypothetical protein and porin genes identified from targetedmutagenesis in the pre- vious section were also investigated in this comparative transcriptomics analysis. Out of the 3 hypothetical pro- tein targets (Synpcc7942_1655, Synpcc7942_0122, and Synpcc7942_0900), Synpcc7942_1655 was the only gene without homologs in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. Both Synpcc7942_0122 and Synpcc7942_0900 had homologs in the other cyanobacterial
Until recently, the possibility of recovering knockout lines by conventional reverse genetic approaches (T-DNA, TILLING) for a specific gene has been dictated by chance. The shorter the gene, the lower the probability to hit it with a T-DNA insertion or a mutation. Routine targetedmutagenesis opens up a new dimension in plant biology and should help to generate mutants in previously difficult to access genes, as well as simultaneously mutate multiple loci and generate large deletions [11,13]. The likelihood of targeting a specific genomic locus is probably af- fected by various factors (e.g. chromatin context) but Cas9 does not seem to be affected by DNA methylation, at least in human cells .
The efficiency of homologous recombination relies on the lenght of the homology arms and the chromatin compactation of the locus. In ES cells the immuoglobulin locu is compacted. Thus, the homologous recombination events occur at a very low frequency. A high number of ES cell colonies have to be analysed to detect a homologous recombination event. Here we describe a reliable and rapid protocol for the analysis of targetedmutagenesis in the immunoglobulin locus using Southern Blot analysis.
In order to detect mutations, enrichment by restriction enzyme digestion was applied in the experiments described in the previous paragraph. In order to compare the efficiency of targetedmutagenesis after NCas9F protein delivery with that after T-DNA transfer directly, amplicon deep sequencing was performed. Amplicons were generated by PCR from DNA isolated from nine independently infiltrated leaves, 10 days after inoculation. These PCR amplicons covering the protospacer and PAM sequences were sequenced using the illumina HiSeq 2500 platform. Figure 2 depicts the results as relative numbers of mutated reads per position. Most deletions detected were found 3 to 13 bp upstream of the PAM sequence irrespective of how Cas9 was delivered (Fig. 2a). Deletions in cells expressing Cas9 and sgRNA from a single T-DNA were likewise found 3 to 13 bp upstream of the PAM, but at a higher frequency than in cells into which the NCas9F protein had been translocated (Fig. 2a). Almost all insertions found with both translocated NCas9F and Cas9 and sgRNA expressed from a single T-DNA were located 3 to 4 bp upstream of the PAM (Fig. 2b). Such single bp insertions were not detected in sequenced amplicons obtained from non-infiltrated N. benthamiana tissue. The percentage of sequences with a deletion obtained from tissue with translocated NCas9F was found to be 0.24%, but the percentage seen after expression of Cas9 and sgRNA expressed from a single T-DNA was found to be eighteen fold higher at 4.31%. Similarly, the insertion frequency with translocated NCas9F was found to be 1.55% and 12 fold higher at 19.8% after expression of Cas9 and sgRNA from a single T-DNA. Further analysis revealed that around ~80% of these single bp insertions are adenosine or thymidine insertions.
Fig. 1. Efficient targetedmutagenesis of EGFP in Tg( CAGG:EGFP ) transgenic axolotls. (A) Distribution of phenotypes in axolotl (Ambystoma mexicanum) embryos from a mating between a wild-type and hemizygous Tg(CAGG:EGFP) animal injected with various concentrations of cas9 mRNA and an EGFP-directed sgRNA at 3 weeks post-injection, as assessed by fluorescence microscopy. Whereas ∼ 50% of embryos injected with lower concentrations of cas9 and sgRNA displayed normal EGFP expression, those injected with the highest concentrations of cas9 and sgRNA displayed mosaic EGFP expression. Any animal exhibiting clones of EGFP-negative cells was classified as ‘ partial EGFP ’ . The survival rate in all cas9 and sgRNA-injected embryos did not differ from that of embryos injected with nls-EGFP mRNA only (right column). (B-E 0 ) Whereas 6-month-old uninjected Tg(CAGG:EGFP) animals display strong uniform EGFP expression in both their heads (B 0 ) and tails (C 0 ), siblings injected with EGFP-directed sgRNA and cas9 (D,E) display a dramatic loss of EGFP, with one individual with only EGFP-positive cells apparent in the head (D 0 ) and another with EGFP expression only in the tail (E 0 ). (B-E) Brightfield; (B 0 -E 0 ) EGFP fluorescence. (F) All 34 sequences of cloned PCR products of the EGFP locus in a single animal injected with an EGFP-directed sgRNA and cas9 that displayed no apparent EGFP expression contain indels at the targeted site (yellow). The size of each deletion ( – ) or insertion (+; in red) and frequency of occurrence among clones are indicated.
We recently reported that TALEN target sites are distrib- uted in the mouse genome at an average spacing of 14 bp, enabling genome-wide targetedmutagenesis at high pre- cision. In particular, we provided proof-of-principle that TALENs and oligodeoxynucleotides (ODNs) can be applied in one-cell embryos to introduce targeted mutations (Wefers et al. 2013). For HR- and NHEJ-mediated gene modiﬁcations, we achieved rates of 2% and 6%, respectively, using experi- mental conditions that were not yet optimized. Higher rates of NHEJ-mediated nucleotide deletions (.40%) were obtained upon the microinjection of TALEN mRNAs into the cytoplasm of one-cell embryos, tolerating larger injec- tion volumes (Sung et al. 2013). Nevertheless, for the crea- tion of targeted mutations it is instrumental to deliver DNA templates for HR together with TALEN mRNAs directly into the pronucleus, tolerating only minimal injection volumes. To set up an efﬁcient routine procedure for mutagenesis we enhanced the activity of TALEN mRNAs to optimize nuclease expression upon pronuclear delivery, such that one or more knockin or knockout alleles are obtained among a group of mice derived from a single microinjection experiment. Upon the establishment of a mutant by embryo manipulation, the genotyping of breeding colonies imposes a constant work- load. PCR-based protocols for the detection of subtle muta- tions often require the digestion of PCR products and gel electrophoresis. To minimize these efforts we validated whether high-resolution melt analysis (HRMA) represents a reliable and simpliﬁed tool for the genotyping of mouse mutants. HRMA identiﬁes mutant PCR products by their spe- ciﬁc denaturation proﬁle (Liew et al. 2004) and requires no restriction digestion and size separation of PCR products.
Results: Here, two different TALEN techniques, using custom and self-assembled TALEN constructs, were applied and compared. The MpNOP1 gene was selected as a candidate gene, as the respective knockout mutant has been shown to lack air chamber formation, representing an easily traceable phenotype. We demonstrate that both TALEN approaches are successful in Marchantia yielding high gene targeting efficiencies of over 20%. Investigation of selected G1 up to G4 generations proved the stability of the knockout mutants. In 392 analyzed T1 plants, no addi- tional phenotypes were observed and only one chimeric knockout plant was detected after an extended cultivation period. Interestingly, two out of the 24 sequenced mutants harbored indels causing in-frame mutations and revealed novel Mpnop1-related phenotypes. This demonstrates the potential to detect crucial amino acids and motives of targeted proteins, which is of special interest for essential genes where full knockouts are lethal. The FastTALE ™ TALEN assembly kit enables the rapid assembly and ligation of the TALEN arms within half a day. For transformations, custom and assembled constructs were subcloned into Marchantia binary vectors possessing the MpEF1α promoter.
In conclusion, our study of the HPV16 E2 protein by alanine substitution mutagenesis of conserved amino acid residues in the N terminus has provided evidence that E2 is a multifunc- tional protein whose different activities can be separated. The strong transcriptional activation function of E2 can be disso- ciated from its ability to enhance E1-mediated, origin-specific DNA replication. The DNA replication function of E2 appears to depend on its ability to bind E1. Furthermore, although some of the stable mutants were not capable of strong trans- activation, they still manifested a weak transactivation function in C33A cells which was revealed by using a reporter construct containing the HPV16 LCR as an enhancer element. This result indicates that even the transcriptional regulatory prop- erties of E2 may be multifunctional and dependent on specific cellular factors. The E2 mutants described in this report should prove useful in probing the different activities of E2 and in the identification of the specific cellular factors associated with these various functions.
Background: Sickle cell disease (or simply, SCD) is an inherited hemoglobinopathy which is mostly prevalent among persons of African descent. SCD results from a monogenic (Hemoglobin, beta) point-mutation (substitution of the base Adenine with Thymine at position six) that leads to replacement of the amino acid glutamic acid (E) with valine (V). Management of SCD within resource-poor settings is largely syndromic, since the option of cure offered by bone-marrow transplantation (BMT) is risky and unaffordable by most affected individuals. Despite previous reports of repair and inhibition of the sickle beta-globin gene and messenger ribonucleic acids (mRNAs), respectively in erythrocyte precursor cells via gene-targeting using an oligomer-restriction enzyme construct and either ribozyme- or RNA-DNA chimeric oligonucleotides (or simply third strand binding), gene-therapy to treat SCD still remains largely preclinical. In the wake of the advances in target- gene- mutagenesis and repair wrought by zinc finger nuclease (ZFN) technology, it was hypothesized that SCD may be cured by the same. The goal of this study thus, was constructing a database of zinc finger arrays (ZFAs) and engineering ZFNs, that respectively bind and cleave within or around specific sequences in the sickle hemoglobin, beta (−β S
Genome editing technologies enable precise modifica- tion of DNA sequences in vivo and promise a novel revo- lution in crop improvement (Sun et al. 2016; Feng et al. 2013). The clustered regularly interspaced short palin- dromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system has revolutionized genome editing and become widely popular because of its specificity, simpli- city, and versatility. It allows targeted genome editing in organisms guided by a customizable small noncoding RNA called single guide RNA (sgRNA). Once susceptibi- lity genes targeted by TAL effectors have been identified, the CRISPR/Cas9-mediated genome editing strategy can be employed to create a target mutation in the susceptibi- lity genes. Although it was not edited by the CRISPR/ Cas9, Os11N3 (also known as OsSWEET14), the suscepti- bility gene targeted by AvrXa7 and PthXo3, has been edited by Transcription Activator-Like Effector Nucleases (TALENs) to create bacterial blight-resistant rice through disrupting the EBE site in the promoter region (Li et al. 2012; Blanvillain-Baufume et al. 2017). It can also be applied to negative regulators of disease resistance that have been studied for the last decades (Grand et al. 2012; Wang et al. 2015; Chern et al. 2005). However, to date, only a few examples of improvement of disease resistance using the CRISPR/Cas9 approach have been reported (Wang et al. 2016; Pyott et al. 2016; Peng et al. 2017). For Os8N3, studies on its knockdown rice plants using the gene silencing system and promoter mutations reported that they showed enhanced resistance to Xoo while dis- playing abnormal pollen development (Yang et al. 2006; Chu et al. 2006). Recently, CRISPR/Cas9-mediated knock- out of Os8N3 displayed decreased sucrose concentration in the embryo sacs and defective grain filling, suggesting that Os8N3 plays important role in sucrose transport during early stage of rice grain filling (Ma et al. 2017; Yang et al. 2018).
amplified using Pfu polymerase (Stratagene) and specific primers with designed restriction enzyme recognition sequences. The region of PRD1 genome (nucle- otide coordinates 4894 to 6309 ) containing the structural genes XXXI and V was amplified using the viral DNA as a template. The fragment was purified from low-melting-point agarose gel and ligated into pSU18 vector under the lac promoter, resulting in plasmid pJB500. Using pJB500 as a template, a BamHI site was generated using the QuikChange site-directed mutagenesis kit (Strat- agene) at the beginning of gene V (position 5305 in the PRD1 genome), resulting in plasmid pJB501. Chromosomal DNA from Escherichia coli JE2571 (14), carrying the wild-type ␤-galactosidase gene, was isolated and used as a template to amplify the lacZ␣ fragment. The fragment was cloned into the BamHI site of pJB501, and the resulting plasmid was named pJB504. Using plasmid pJB500, carrying the wild-type gene V, as a template the in vitro mutagenesis method was used to introduce an amber mutation into the beginning of gene V (5299) corresponding to the Q4 residue in protein P5. The resulting plasmid was named pJB515.
To examine the subcellular localization of ptTES1, we fused the full-length amino acids to eGFP. P. tricornutum transformants expressing the ptTES1-eGFP construct showed GFP fluorescence mainly in the plastid (Fig. 2), where de novo fatty acid synthesis takes place. Further- more, we performed cell fractionation and western blot with α-ptTES1 antibody. The native ptTES1 protein could be detected in the plastid as well as in the cyto- sol (Additional file 2: Figure S2). The GFP fluorescence detection and immunoblot result do not correlate with in silico predictions of subcellular localization. Indeed, there is no evidence of a bipartite transit peptide made of a signal peptide , followed by a chloroplast-transit peptide , nor of an ASAFAP motif involved in plas- tid targeting in Phaeodactylum . Rather, pTES1 was predicted to be targeted to the mitochondrion, based on the Mitoprot predictive tool . Although P. tri- cornutum ptTES1 was identified to have acyl-CoA thi- oesterase activity in vitro as purified protein, whether it is directly involved in the hydrolysis of acyl-CoAs in the plastid in vivo remains unknown. To determine whether inactivation of ptTES1 has an effect on fatty acid and
The inability to produce a significant number of excon- jugants with constructs containing cas9 suggests that the gene is toxic in Synechococcus 2973 when it is introduced on a medium copy number plasmid. The fact that only five colonies were yielded from conjugation with a con- struct in which the cas9 RBS was removed (compared to the ~250 colonies with the construct lacking cas9) sug- gests that these exconjugants are “escapees” with respect to cas9 toxicity. Furthermore, we conclude that the cas9 gene cannot be stably maintained in Synechococcus at a medium copy number level. Although the reason behind Cas9 toxicity is currently unclear, one possibility is that S. pyogenes Cas9 has off-target effects in cyanobacte- rial cells. The enzyme may be cleaving genomic DNA in regions other than those targeted by the synthetic sgRNA, and that the cell is unable to repair these breaks, thus resulting in lethality.
C URRENT methods to generate mutations in the genome of Caenorhabditis elegans, including chemical mutagen- esis and imprecise excision of transposons, all rely on recovering mutations in large-scale mutagenesis screens. Re- cently, several groups reported the use of the Streptococcus pyogenes CRISPR/Cas9 system to generate double-strand break (DSB)-induced mutations in speciﬁc genomic loci in model systems including yeast (Dicarlo et al. 2013), ﬂies (Bassett et al. 2013; Gratz et al. 2013; Yu et al. 2013), mammalian cells (Cho et al. 2013a; Mali et al. 2013), and zebraﬁsh (Hwang et al. 2013). Because of the enormous potential for targeted genome engineering, we here investi- gate the suitability of the CRISPR/Cas9 system for use in C. elegans. This article is one of six companion articles in this issue (Chiu et al. 2013; Cho et al. 2013b; Katic and Grosshans 2013; Lo et al. 2013; Tzur et al. 2013) that present different approaches to and features of Cas9-CRISPR genome editing in C. elegans.
The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often ex- ceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cyto- megalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, marker- less deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral ge- nome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates.