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Cas proteins: dodgy scaffolding in breast cancer

Cas proteins: dodgy scaffolding in breast cancer

transduction complexes in response to several stimuli, such as growth factors, hormones and extracellular matrix components. Given their ability to integrate and coordinate multiple signalling events, Cas proteins have emerged as crucial players in the control of mammary cell proliferation, survival and differentiation. More importantly, it has been found that alterations of their expression levels result in aberrant signalling cascades, which promote initiation and progression of breast cancer. Based on the increasing data from in vitro, mouse model and clinical studies, in this review we will focus on two Cas proteins, p130Cas/BCAR1 and Nedd9, and their coupled signalling pathways, to examine their role in mammary cell transformation and in the acquirement of invasiveness and drug resistance of breast cancer cells.

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CRISPR-Cas Systems in Lactic Acid Bacteria.

CRISPR-Cas Systems in Lactic Acid Bacteria.

Clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR- associated (cas) genes constitute the CRISPR-Cas adaptive immune systems in bacteria and archaea (1). CRISPR loci are identified in genomes by locating regions of conserved repeats interspersed by short variable sequences, spacers, that are adjacent to cas genes. The immune function of the systems is carried out by Cas proteins in three steps: first, the acquisition of DNA from an invader is stored between conserved repeats; next, the entire repeat-spacer array is transcribed into a single RNA transcript that must be processed into individual small interfering units during the expression phase; and finally, during interference, the guide RNAs direct Cas proteins to its target got cleavage of foreign invaders in a highly sequence-specific manner upon reinfection (2-6). Repeat-spacer arrays record the immunization timeline of an organism as spacer sequences, which are always added to the array at the leader end; this can provide a unique, hypervariable locus that can be used to genotype organisms so as to provide insights into their phylogenetic divergence and relatedness to other strains (7). Identifying the source of the spacer sequences, called the protospacer sequence, can tell a story of host-prey dynamics in environments where phage and plasmid intrusions are common and often lethal (8).

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Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems

Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems

These predictions attracted the attention of several research groups resulting in considerable experimental support for the above hypothesis [6]. These studies have elucidated many important details of the molecular mechanisms of the CRISPR-Cas systems and the three distinct functional stages of their operation [7,8]. During the first stage, adaptation, short pieces of DNA (charac- teristic length of approximately 30 bp) homologous to virus or plasmid sequences (known as proto-spacers) are integrated into the CRISPR loci [6,9,10]. The short (3 or 4 nucleotides) proto-spacer adjacent motifs (PAMs) located immediately downstream of the proto-spacer appear to determine the selection of the protospacer fol- lowed by integration into a pre-existing CRISPR array [11,12]. The second stage, expression and processing, involves transcription and cleavage of long primary tran- script of a CRISPR locus (pre-crRNA) that is processed into short crRNAs. This step is catalyzed by endoribonu- cleases encoded by the cas genes that either operate as a subunit of a larger complex (e.g. Cascade, CRISPR-asso- ciated complex for antiviral defense in Escherichia coli [13]) or as a stand-alone enzyme, e.g., Cas6 in the archaeon Pyrococcus furiosus [14,15]. At the third stage, interference, the alien nucleic acid (DNA or RNA) is tar- geted by a ribonucleoprotein complex containing a crRNA guide and a set of Cas proteins, and cleaved within or in the vicinity of the PAM sequence [7,9,10,16]. In several CRISPR-Cas systems, crRNA have been shown to be complementary to either strand of the phage or plasmid which is best compatible with DNA being the target [6,17]. Direct demonstration of DNA being the tar- get of the CRISPR-Cas machinery has come from experi- ments in Staphylococcus epidermidis. In this case, insertion of a self-splicing intron into the proto-spacer sequence of the target gene rendered the respective plas- mid resistant to the CRISPR-mediated immunity [18]. Recently, the E. coli Cascade complex containing crRNA has been shown to recognize the target DNA, with the specificity defined by the crRNA sequence, and displace the non-complementary strand in an energy-independent manner [8]. However, in vitro experiments with one of the CRISPR-Cas systems (Type IIIB, formerly known as Cmr system or RAMP module) from the archaeon P. fur- iosus showed that the crRNA rather targets the mRNA [15]; it remains to be determined whether this is also the case in vivo or the P. furiosus CRISPR-Cas systems target alien DNA in addition to or instead of mRNA. In any

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Özcan, Ahsen
  

(2019):


	Characterization of the type IV CRISPR-Cas system of aromatoleum aromaticum EbN1.


Dissertation, LMU München: Fakultät für Biologie

Özcan, Ahsen (2019): Characterization of the type IV CRISPR-Cas system of aromatoleum aromaticum EbN1. Dissertation, LMU München: Fakultät für Biologie

The cas genes and a minimal CRISPR array of the A. aromaticum EbN1 (type IV CRISPR- Cas system) were transferred into Escherichia coli BL21 AI to uncover the RNA and protein components of this CRISPR-Cas type. Type IV crRNAs were shown to yield unusually short 7 nucleotide 5′-repeat tags and stable 3′ hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR– ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained and the active site of the enzyme was determined. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified the cRNPs’ protein–protein and protein–RNA contact sites. A possible PAM-sequence dependent DNA targeting mechanism of this complex and the involvement of a type IV CRISPR-Cas associated DinG helicase are discussed.

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CRISPR-Cas Systems for Diagnosing Infectious Diseases

CRISPR-Cas Systems for Diagnosing Infectious Diseases

Abstract: Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas9, Cas12, Cas13, Cas14) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, or lateral flow assay detection. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing coronoviral nCov-2019 infection) urgently need the developing of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.

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Multidrug Resistant Enterococci Lack CRISPR cas

Multidrug Resistant Enterococci Lack CRISPR cas

volunteers (Com12 and Com15). All were isolated in the early to mid-2000s. We identified an E. faecium CRISPR-cas locus (EfmCRISPR1-cas) in three genomes (Fig. 4). This locus is found between homologues of EfaeDRAFT_1858 and EfaeDRAFT_1857 from the previously sequenced clinical isolate E. faecium DO draft genome (GenBank accession no. ACIY00000000), which itself lacks CRISPR (25). EfmCRISPR1- cas possesses a palindromic repeat sequence that is divergent from those of the E. faecalis CRISPR-cas loci (see Fig. S1 in the supplemental material). The four predicted Cas proteins encoded by EfmCRISPR1-cas are consistent with the Nmeni subtype (Table S4). However, EfmCRISPR-cas differs from the E. faecalis Nmeni subtype by the presence of an ORF of unknown function 5= to the csn1 gene which is conserved and unique to the three strains possessing EfmCRISPR-cas (Fig. 4). A Blastn comparison of the 20 EfmCRISPR1-cas spacers to sequences in the NCBI nonredun- dant nucleotide database reveals identity between two spacers and previously identified clostridial and lactococcal phages (Table 2). MLST phylogeny and acquired antibiotic resistance profiles were determined for the eight E. faecium strains. Four strains be- long to the hospital-adapted CC17, being either ST17 or double- locus variants of ST17 (Fig. 5; see Table S5 in the supplemental material). As expected for CC17 strains (1), all are MDR. Three of the CC17 strains possess vanA, encoding vancomycin resistance, while the fourth CC17 strain constitutes a novel sequence type variant and lacks vanA. The remaining four strains belong to other sequence types and lack acquired antibiotic resistance genes (Fig. 5). Although the E. faecium analysis was smaller in sample size than that for E. faecalis, the distribution of EfmCRISPR1-cas in these genomes supports the conclusion that MDR enterococcal strains generally lack CRISPR-cas; in this case, three of four MDR strains lack EfmCRISPR1-cas, and these three strains are vancomycin-resistant members of the hospital-adapted CC17 lin- eage.

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CRISPR-Cas Targeting and Escape in Lactobacillus gasseri.

CRISPR-Cas Targeting and Escape in Lactobacillus gasseri.

Due to prokaryotes often lacking robust endogenous DNA repair pathways, double- stranded cleavage of bacterial genomes via CRISPR-Cas is often deadly to the cell (Selle & Barrangou, 2015b). Although this lethality presents several interesting challenges in the arena of bacterial genome editing, it also allows for the use of CRISPR-Cas as an “antimicrobial.” Essentially, CRISPR-Cas systems can be co-opted to enact lethal damage on particular bacterial populations through sequence-specific self-targeting of the genome, leading to the reduction of undesirable environmental populations. If a strain that contains a CRISPR-Cas system is provided with a self-targeting spacer sequence, CRISPR-Cas will destroy its own host’s genome. The lethal effect of CRISPR targeting on bacterial genomes was first observed in 2013, showing that self-targeting of the host chromosome led to a non- reversible ~10 5 reduction in viable counts (Vercoe et al. 2013). There are two strategies to using CRISPR-Cas to specifically target the genomes of particular strains, and they depend on whether or not the strain intended for elimination contains a native CRISPR-Cas system. If the strain of interest contains a native CRISPR-Cas system, it can be eliminated through simply providing the cell with a plasmid that contains guide RNA with spacer sequence that matches a unique protospacer located on the bacteria’s genome. If the strain of interest does not contain a native CRISPR-Cas system, a plasmid must be provided that not only includes the synthetic guide but also the Cas proteins necessary for interference.

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Influence of aging on medial olivocochlear system function

Influence of aging on medial olivocochlear system function

medial efferent pathways; in that study, the tests included sub- jects of different ages with normal hearing thresholds on tonal audiometry. Therefore, the influence of perceptive hypoacusis on MOC system function was excluded. Testing the suppres- sive effect in different age subgroups, they showed (similar to our article) that significantly weaker suppression of OAE level with CAS occurs in older subjects than in younger ones (MOC suppression for CEOAEs: subgroup of 20–39 years, contralateral stimulation, - 2.2 dB; subgroup of 60–79 years, contralateral stimulation, - 0.5 dB; 19 and for DPOAEs, whole

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Evaluation of the Sefton Community Adolescent Service (CAS)

Evaluation of the Sefton Community Adolescent Service (CAS)

Finally, the evaluation has underlined the critical importance of setting in place a robust performance management system for the CAS. The work undertaken latterly during the pilot, to extract data from the LSC children’s record, was commendable and provided insights to case status, progress, and (some) destinations data. Going forwards, however, there is a real priority to establish a system up-front for the CAS that reflects the metrics on the score-card, and allows for the disaggregation of data held on individual young people within the cohort. If the CAS is wider than a social care intervention, then a more effective mechanism is needed to monitor and review the status of young people supported by CAS according to education and employment, health, housing, social care, crime and antisocial behaviour. There might be potential to draw upon the learning from the Troubled Families service in Sefton to help scope a suitable model for the CAS service.

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Aucune fois, un cas de grammaticalisation ?

Aucune fois, un cas de grammaticalisation ?

Résumé . Fois apparaît dans de nombreuses constructions avec une distinction majeure se résumant dans deux types d’emplois : un emploi nominal et un emploi grammatical. La double fonctionnalité de fois nous pousse à nous interroger sur son statut dans notre objet d’étude aucune fois : emploi nominal ou grammatical ? Ce qui nous amène à la question de sa grammaticalisation. Pour tenter de résoudre cette question, nous traiterons, à travers son évolution diachronique, ses caractéristiques graphiques, morphosyntaxiques et sémantiques. Au plan graphique, aucune fois se caractérise par la variation de son emploi en un seul mot ou en deux mots. Au plan morphosyntaxique, nous citerons les cas du pluriel (aucunesfois/aucunes fois), les cas d’insertion d’indéfini entre aucune et fois (aucune autre fois). Au plan sémantique, aucune fois, mis à part un exemple dans une phrase négative avec la traduction moderne de « zéro fois », est doté essentiellement d’une valeur positive au sens moderne de « parfois/quelquefois » ou de « une fois » suite à son emploi dans des phrases positives.

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Do calculus students demand technology integration into learning environment? case of instructional differences

Do calculus students demand technology integration into learning environment? case of instructional differences

The interviews have been used to evaluate how the outputs of the teaching process change according to thinking types. Semi-structured interviews were conducted with the four selected students and were audio recorded. Describing who will be selected for the interview and the principles that affect the selection will make it easy to interpret the findings. Considering the fact that along with types of thinking, differences in teaching environments might also affect the demands of students regarding technology; students who have sufficied to represent these differences were chosen. For this reason, participants in the interviews were selected using a purposeful sampling technique from voluntary students. The main selection criteria were that each participant had a different thinking type in each environment (CAS or Traditional). Interviews were con- ducted with visual and analytical thinkers (contrasting cases) in order to determine the similarities and differences between the technological demands of students (Haciomer- oglu et al. 2010). Participants were coded with a formulization in the form of environ- ment and thinking types. Analytic participants who are in traditional and CAS- supported environments were coded with Tra-A and CAS-A, respectively. Also, Tra-V and CAS-V codes were used to define visual participants who are in traditional and CAS-supported environments. MPI points (out of 36) of the participants are shown below: CAS-A was 7, CAS-V was 25, Tra-A was 5 and Tra-V was 22. Each participant’s interview lasted approximately 40 min, and each of them was transcribed. During inter- views, participants were asked to explain their attitudes towards the usage of CAS in calculus considering their expectations of the course. Interview data were analyzed with an open coding method, which consists of identifying, naming, categorizing, and de- scribing phenomena found in the text for the thematic analysis (Strauss & Corbin, 1998). During the open coding process, semi-structured interview data were tran- scribed, and responses to the same interview questions of the participants who had dif- ferent thinking types and settings were coded in terms of previously identified categories. The findings obtained by usage of frequently-repeated objectives and acqui- sitions in the interviews were summarized in the table (Table 2). Some selected quotes (for each participant) were also shared in the text to be able to characterize the partici- pants’ attitudes towards usage of CAS.

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Phylogenetic Distribution of CRISPR Cas Systems in Antibiotic Resistant Pseudomonas aeruginosa

Phylogenetic Distribution of CRISPR Cas Systems in Antibiotic Resistant Pseudomonas aeruginosa

As another focused example of the plethora of interesting CRISPR-Cas-genomic island interactions observed in our data set (see Table S4 in the supplemental material), we looked closely at a widely distributed spacer targeting the pilV2 gene of the conjuga- tive type IVb pilus of pKLC102, PAPI-1, and PAGI-5. While we identified multiple spacers in many strains targeting these three widely distributed conjugative elements (Fig. 5B), we chose to focus on a single spacer targeting pilV2. PilV2 is the minor subunit of the pilus filament and is required for functional conjugation- mediated horizontal gene transfer of these and other important accessory genomic elements (58). As pilV2 is required for function and is highly conserved between similar conjugative elements, it provides both a perfect target for the CRISPR-Cas bacterial im- mune system and an excellent marker for determining the carriage rate of similar elements in our clinical library. Roughly 40% of clinical isolates within our library contained conjugative elements harboring a 100% conserved target and protospacer-adjacent mo- tif (PAM) sequence within the pilV2 gene, clearly indicating the ubiquity of these elements in the P. aeruginosa global population. The diversity of genetic elements potentially targeted by P. aeruginosa CRISPR-Cas systems is striking. However, well char- acterized mobile genetic elements account for only ~30% of unique spacer matches, while 55% are complementary to largely uncharacterized portions of the P. aeruginosa accessory genome. These data clearly illustrate the need for more accurate curation of P. aeruginosa accessory genome elements, a challenging process due to the chimeric nature of many mobile elements and the enor- mous number of P. aeruginosa strains being sequenced. However, as this study establishes a massive library of spacer sequences to highlight these interesting elements within NGS data, this chal- lenge should be more manageable.

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CAS Based Approach for Automatic Data Integration

CAS Based Approach for Automatic Data Integration

Many attempts to constrain variety and address se- mantic heterogeneity have been proposed, including on- tology based solutions. Elements of a given data structure are mapped to an ontology for aiding with semantic re- solution tasks. However, ontologies themselves are not constrained. Thousands of competing ontologies have been built and published in recent years [26-30]. Further, several non-compatible ontology mechanisms have been proposed and put to use [10-13]. From a CAS perspec- tive, these were attempts to build a regulator. As explain- ed earlier, they too failed to deliver the promise, since they did not manage to control semantic variety and rela- tions variety. These solutions also have the risk of creat- ing a circular reference (Figure 1), thus not leading to a resolution and not meeting their proposed goals.

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CONSTRUCTION AND DEVELOPMENT OF COMPUTER ATTITUDE SCALE  (CAS)

CONSTRUCTION AND DEVELOPMENT OF COMPUTER ATTITUDE SCALE (CAS)

The present scale (CAS) has been developed with an objective of assessing teacher‟s attitude towards the use of computer in teaching. The investigator had gone through different scales before constructing and developing the scale. Computer Attitude Scale by B. Yushau(‎2006) for assessing teachers' attitudes toward computers, Computer Attitude Scaleby R.C. Kluever (‎1992)and (1994) for students, Computer Attitude Scalefor language teachersby N.M. Daud(1995 ), Computer Attitude Scale (CAS) by Loyd and Gressard (1984), Computer Attitude Scale for Teachers (CAST) by Yuen and Ma (2001); observed that majority of scales were developed for students and in foreign conditions. So there is a need for developing a scale which is applicable in Indian conditions. The tool would help to assess the attitude of teachers towards the use of computer. Moreover with the changing times, the situations also change. The tool in use needs to be revised or developed accordingly.

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ANC experiments for nuclear astrophysics in NPI CAS

ANC experiments for nuclear astrophysics in NPI CAS

Abstract. Asymptotic Normalization Coefficients method is one of the indirect methods used in nuclear astrophysics. The method allows to deduce a direct part of a radiative capture cross section from the measurement of a two body reaction under specific con- ditions. The experimental works started in collaboration of NPI CAS, TAMU and INFN LNS from 90-ties and continue till the present. The introduction to this method is pre- sented and experimental experiences from NPI CAS and the collaboration are shared.

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Solubility of CaS in CaCl2 LiCl Eutectic Melt

Solubility of CaS in CaCl2 LiCl Eutectic Melt

The charge in the crucible was halved from 300 g to 150 g in order to shorten the depth of melt (55 mm when 300 g is charged) and to be homogenized in a shorter period. Figure 6 shows the results using 150 g melt. The depth is estimated as 23 mm. Assuming the solubility limit locates near 0.2­ 0.3 mol % CaS, the added concentration of CaS was set 0.4 mol%. The analyzed concentrations from the quenched samples did not arrive at the added concentration, and they converged to 0.31 « 0.05 mol%CaS at 973 K, and 0.22 « 0.05 mol% at 873 K. The authors would like to report these values are true saturation limits of this melt.

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CAS Complications Rate and Relation to Risk Factors

CAS Complications Rate and Relation to Risk Factors

In this prospective clinical study, we have focused our attention on the risk factors and their relationship to the occurrence of serious early and late complications. So far, a higher age was the single documented risk factor that had a significant impact on the incidence of serious complications. We rely on the criteria of the SPACE study [14], while dividing patients into the age groups (a group under 75 years and a group more than 75 years inclusive), primarily because of the better comparability and interpretation of results [11]. As in many published works [7-12,15,16], higher age had, at the marginal significance level, an impact on the incidence of TIA and early death. Higher age also had a significant impact on the incidence of restenosis, and, therefore, understand- ably the group of elderly patients had a higher overall mortality rate. Bonati et al. state that CAS should be avoided in older patients [17]. We concluded that higher age is a relative contraindication to the intervention.

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Organizational interventions employing principles of complexity science have improved outcomes for patients with Type II diabetes

Organizational interventions employing principles of complexity science have improved outcomes for patients with Type II diabetes

do not improve process or outcome measures for patients with the chronic illness diabetes [3-5]. Only 50% of rand- omized or controlled clinical trials meeting criteria for this systematic review demonstrated significant improve- ment in most or all endpoints. In support of our hypoth- esis that implementation strategies consistent with CAS theory will be more likely to be effective, we found a sig- nificant relationship between the number of CAS charac- teristics utilized in an intervention and the intervention's effectiveness in improving process or outcome measures in patients with Type II diabetes. This was true in studies conducted across a diversity of clinical settings and reported outcomes. This finding potentially widens the scope of the application of CAS theory beyond the current literature that focuses on describing clinical settings as CAS [14,15], to build upon the idea that we can use CAS to both design and implement interventions [16,20] that are more likely to lead to improved performance.

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CRISPR Cpf1 proteins: structure, function and implications for genome editing

CRISPR Cpf1 proteins: structure, function and implications for genome editing

Previously, it has been demonstrated that CRISPR– Cas9 could be engineered to modulate gene expression in plants [104]. The T-rich PAM made CRISPR–Cpf1 a useful reagent for targeting plant AT-rich promoter regions as a transcriptional regulator. To this end, Tang et  al. generated dAsCpf1 and dLbCpf1 by inducing mutations in D908A and D832A [12]. Flowingly, they made dCpf1-SRDX fusion proteins as a transcriptional repressor [104]. Under the control of a dual ubiquitin promoter system, the CRISPR–dCpf1–SRDX repres- sors were transfected into the Arabidopsis. The pur- pose of these transcriptional repressors was to target the promoter of a noncoding RNA, i.e., miR159b. Both dAsCpf1-SRDX and dLbCpf1–SRDX repressors down-regulated the expression of miR159b less than 10% of the wild-type in randomly chosen T1 trans- genic lines. Albeit, a more variation was seen in the case of dLbCpf1–SRDX. These findings suggested that although the nuclease activity of AsCpf1 was less than LbCpf1, it was more tightly bound to DNA [105].

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Different genome stability proteins underpin primed
and naïve adaptation in E  coli CRISPR Cas immunity

Different genome stability proteins underpin primed and naïve adaptation in E coli CRISPR Cas immunity

ends of appropriate DNA strands. (A) EMSAs of Cas1 binding to branched DNA substrates. Cas1 monomer protein concentrations are given above the panels, in reactions containing 6 nM of DNA. ‘X’ indicates stable Cas1-DNA complex observed with forks. Nucleotide (nt) lengths of DNA strands in fork substrates are indicted (25 or 50 nt), and 26 nt for the ssDNA flap regions of Fork-1 or Fork-2. (B) EMSA showing the effect of pre-mixing Cas1 with Cas2 on binding to fork-1, at protein monomer concentrations indicated above the panel, in reactions containing 6 nM of DNA. ‘X’ denotes defined Cas1-DNA complex, and ‘Y’ a second complex requiring Cas1 fork binding in the presence of Cas2. Lanes 5–10 show that complex Y is not formed when Cas1 mutant proteins (R84G, R123G or R138G) were pre-incubated with Cas2 as indicated because each is unable to bind to the fork DNA. (C) Urea denaturing gels showing products from nicking of fork DNA (6 nM) by Cas1 (2.5–25 nM). Numbers 26 and 4 refer to the nucleotide length of the ssDNA region on the labeled DNA strand of each fork. (D) Urea denaturing gel comparing product of Cas1 (25 nM) nicking fork-1 (lane 2) to lack of detectable product from any strand of Holliday junction (lanes 3–6) or fully base paired fork (lanes 7 and 8). (E) Urea denaturing gels showing product of Cas1 (25 nM) nicking fork-1a (6 nM, lanes 1 and 2) or fork-1 (lanes 6 and 7) compared to 32 P end labeled marker DNA strands (M1–M3) of known nucleotide

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