CRISPR-Cas9 Genome Engineering: Trends in Medicine and Health.

The ability to engineer biological systems and organisms holds enormous potential for applications across basic science, medicine, and biotechnology. Over the past few decades, the development of CRISPR (clustered regularly interspaced short palindromic repeat) has revolutionized the whole genetic engineering process utilizing the principles of Watson-Crick base pairing. CRISPRCas9 technology offers the simplest, fastest, most versatile, reliable, and precise method of genetic manipulation, thus enabling geneticists and medical researchers to edit parts of the genome by removing, adding, or altering sections of the DNA sequence. The current review focuses on the applications of CRISPR-Cas9 in the field of medical research. Compared with other gene-editing technologies, CRISPR/Cas9 demonstrates numerous advantages for the treatment of various medical conditions, including cancer, hepatitis B, cardiovascular diseases, or even high cholesterol. Given its promising performance, CRISPR/Cas9 gene-editing technology will surely help in the therapy of several disorders while addressing the issues pertaining to the minimization of the off-target effects of gene editing and incomplete matches between sgRNA and genomic DNA by Cas9.

Phylogenetic analysis suggests single and multiple origins of dihydrofolate reductase mutations in Plasmodium vivax.

Pyrimethamine was first introduced for the treatment of malaria in Asia and Africa during the early 1980s, replacing chloroquine, and has become the first line of drugs in many countries. In recent years, development of pyrimethamine resistance in Plasmodium vivax has become a barrier to effective malaria control strategies. Here, we describe the use of meta-barcoded deep amplicon sequencing technology to assess the evolutionary origin of pyrimethamine resistance by analysing the flanking region of dihydrofolate reductase (dhfr) locus. The genetic modelling suggests that 58R and 173L single mutants and 58R/117N double mutants are present on a single lineage; suggesting a single origin of these mutations. The triple mutants (57L/58R/117N, 58R/61M/117N and 58R/117N/173L) share the lineage of 58R/117N, suggesting a common origin. In contrast, the 117N mutant is present on two separate lineages suggesting that there are multiple origins of this mutation. We characterised the allele frequency of the P. vivax dhfr locus. Our results support the view that the single mutation of 117N and double mutations of 58R/117N arise commonly, whereas the single mutation of 173L and triple mutations of 57L/58R/117N, 58R/61M/117N and 58R/117N/173L are less common. Our work will help to inform mitigation strategies for pyrimethamine resistance in P. vivax.

A novel metabarcoded 18S ribosomal DNA sequencing tool for the detection of Plasmodium species in malaria positive patients.

Various PCR based methods have been described for the diagnosis of malaria, but most depend on the use of Plasmodium species-specific probes and primers; hence only the tested species are identified and there is limited available data on the true circulating species diversity. Sensitive diagnostic tools and platforms for their use are needed to detect Plasmodium species in both clinical cases and asymptomatic infections that contribute to disease transmission. We have recently developed for the first time a novel high throughput 'haemoprotobiome' metabarcoded DNA sequencing method and applied it for the quantification of haemoprotozoan parasites (Theleria and Babesia) of livestock. Here, we describe a novel, high throughput method using an Illumina MiSeq platform to demonstrate the proportions of Plasmodium species in metabarcoded DNA samples derived from human malaria patients. Plasmodium falciparum and Plasmodium vivax positive control gDNA was used to prepare mock DNA pools of parasites to evaluate the detection threshold of the assay for each of the two species. The different mock pools demonstrate the accurate detection ability and to show the proportions of each of the species being present. We then applied the assay to malaria-positive human samples to show the species composition of Plasmodium communities in the Punjab province of Pakistan and in the Afghanistan-Pakistan tribal areas. The diagnostic performance of the deep amplicon sequencing method was compared to an immunochromatographic assay that is widely used in the region. The deep amplicon sequencing showed that P. vivax was present in 69.8%, P. falciparum in 29.5% and mixed infection in 0.7% patients examined. The immunochromatographic assay showed that P. vivax was present in 65.6%, P. falciparum in 27.4%, mixed infection 0.7% patients and 6.32% malaria-positive cases were negative in immunochromatographic assay, but positive in the deep amplicon sequencing. Overall, metabarcoded DNA sequencing demonstrates better diagnostic performance, greatly increasing the estimated prevalence of Plasmodium infection. The next-generation sequencing method using metabarcoded DNA has potential applications in the diagnosis, surveillance, treatment, and control of Plasmodium infections, as well as to study the parasite biology.

Synergistic effect of piperonyl butoxide and emamectin benzoate on enzymatic activities in resistant populations of red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae).

Resistance to grain protectants in Tribolium castaneum (Herbst) is a serious threat to international grain trade. Frequent and overdose application of chemical insecticides is becoming a serious health hazard and cause environmental pollution. Resistance management approaches by using various synergists along with novel compounds has become more imperative to increase efficacy of environmentally safe insecticides. We have evaluated piperonyl butoxide (PBO) and emamectin benzoate mixtures for management of resistant field populations of T. castaneum. The collected strains had demonstrated 50 to 200% resistance already developed against emamectin benzoate as compared with deltamethrin susceptible reference strain. The inclusion of PBO along with emamectin significantly reduced this resistance by at least 28% and the LC50 were lowered from 5.12 to 1.9 µg/ml with the highest synergism ration of 2.7 in resistant strain. Enzymatic assays clearly demonstrated that the specific activities of catalase and acetylcholinesterases were significantly decreased at an average of 80% and 60%, respectively, when PBO was included as a synergist at 1:2 ratio with emamectin benzoate. The results highlight the mechanism that renders the field population resistant to emamectin benzoate and suggests the synergistic role of piperonyl butoxide as a potent additive in grain protectants for resistance management.

Expression and Purification of Transmembrane Protein MerE from Mercury-Resistant Bacillus cereus.

Mercury-resistant (HgR) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to 40 µg/ml against mercuric chloride (HgCl2). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51- 100% homology with the corresponding region of the merA gene of already reported mercuryresistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury (Hg0) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing 30 µg/ml of HgCl2 was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

Selective sweep and phylogenetic models for the emergence and spread of pyrimethamine resistance mutations in Plasmodium vivax.

Pyrimethamine resistance is a major concern for the control of human haemoprotozoa, especially Plasmodium species. Currently, there is little understanding of how pyrimethamine resistance developed in Plasmodium vivax in the natural field conditions. Here, we present for the first time evidence of positive selection pressure on a dihydrofolate reductase locus and its consequences on the emergence and the spread of pyrimethamine resistance in P. vivax in the Punjab province of Pakistan. First, we examined the dihydrofolate reductase locus in 38 P. vivax isolates to look for evidence of positive selection pressure in human patients. The S58R (AGA)/S117N (AAC) double mutation was most common, being detected in 10/38 isolates. Single mutation S117N (AAC), I173L (CTT) and S58R (AGA) SNPs were detected in 8/38, 2/38 and 1/38 isolates, respectively. The F57L/I (TTA/ATA) and T61M (ATG) SNPs were not detected in any isolates examined. Although both soft and hard selective sweeps have occurred with striking differences between isolates, there was a predominance of hard sweeps. A single resistance haplotype was present at high frequency in 9/14 isolates, providing a strong evidence for single emergence of resistance by the single mutation, characteristics of hard selective sweeps. In contrast, 5/14 isolates carried multiple resistance haplotypes at high frequencies, providing an evidence of the emergence of resistance by recurrent mutations, characteristics of soft selective sweeps. Our phylogenetic relationship analysis suggests that S58R (AGA)/S117N (AAC) and S117N (AAC) mutations arose multiple times from a single origin and spread to multiple different cities in the Punjab province through gene flow. Interestingly, the I173L (CTT) mutation was present on a single haplotype, suggesting that it arises rarely and has not spread between cities. Our work shows the need for responsible use of existing and new antimicrobial drugs and their combinations, control the movement of infected patients and mosquito vector control strategies.

Lack of fitness costs associated with acetamiprid resistance in Bemisia tabaci (Hemiptera: Aleyrodidae).

Sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is a devastating pest that can cause severe damage to a range of crops by direct feeding and by plant virus transmission. Because of indiscriminate use of insecticides, this whitefly has developed resistance to several insecticides, including neonicotinoids. Our objectives were to determine fitness components affected by acetamiprid resistance in B. tabaci. Assay results showed that selection with acetamiprid had removed heterozygotes from the field population because the survival rate of the resistant population was significantly greater than that of the field population at a very high dose. Comparison of various life traits between the acetamiprid-selected (Aceta-SEL) population and three other populations showed that the numbers of eggs laid by acetamiprid Aceta-SEL population were significantly lower compared with that of other populations but that the proportions of eggs hatched were significantly higher. However, the time taken by nymphal stages of the Aceta-SEL population to develop was significantly higher than that of the susceptible populations. The intrinsic rate of increase, net reproductive rate, mean generation time, and doubling time of Aceta-SEL was significantly higher than Lab-PK and UNSEL populations, but the growth index was similar for all populations. The growth index and high intrinsic value of Aceta-SEL population suggest that the resistance allele may not have detrimental impact. The lack of fitness costs in B. tabaci could promote the rapid development of resistance to acetamiprid and other neonicotinoids. This resistance could threaten the sustainability of whitefly management program on genetically engineered cotton (Gossypium hirsutum L.) where neonicotinoids are being sprayed to manage sucking pests in the field.

Generating susceptible strain and resistance status of field populations of Spodoptera exigua (Lepidoptera: Noctuidae) against some conventional and new chemistry insecticides in Pakistan.

Two field populations of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) from Dera Ghazi Khan (D. G. Khan) and Multan, Pakistan, were tested for resistance to the 10 most commonly used insecticides in Pakistan by using a standard leaf disc bioassay on the F1 progeny. For comparison, a susceptible strain was generated from the Multan strain, which displayed lower LC50 values for most of the insecticides, by either mass rearing without exposure to insecticides or single-pair crosses against selected insecticides. The single-pair crosses generated a more susceptible strain than mass rearing. The D. G. Khan field strain was highly resistant to cypermethrin, profenofos, spinosad, abamectin, and chlorpyrifos and moderately resistant to deltamethrin, indoxacarb, and methoxyfenozide compared with susceptible lab strain. The Multan strain was highly resistant to profenofos and indoxacarb. Both field populations were susceptible to emamectin benzoate and lufenuron. Rotating these two insecticides with others that show very low, low, or moderate levels of resistance and have different modes of action may be useful for the effective management of this pest.

Cross-resistance, mode of inheritance and stability of resistance to emamectin in Spodoptera litura (Lepidoptera: Noctuidae).

BACKGROUND: Spodoptera litura (F.) is a cosmopolitan pest that has developed resistance to several insecticides. The aim of the present study was to establish whether an emamectin-selected (Ema-SEL) population could render cross-resistance to other insecticides, and to investigate the genetics of resistance. RESULTS: Bioassays at G(1) gave resistance ratios (RRs) of 80-, 2980-, 3050- and 2800-fold for emamectin, abamectin, indoxacarb and acetamiprid, respectively, compared with a laboratory susceptible population Lab-PK. After three rounds of selection, resistance to emamectin in Ema-SEL increased significantly, with RRs of 730-fold and 13-fold compared with the Lab-PK and unselected (UNSEL) population respectively. Further studies revealed that three generations were required for a tenfold increase in resistance to emamectin. Resistance to abamectin, indoxacarb, acetamiprid and emamectin in UNSEL declined significantly compared with the field population at G(1). Furthermore, selection with emamectin reduced resistance to abamectin, indoxacarb and acetamiprid on a par with UNSEL. Crosses between Ema-SEL and Lab-PK indicated autosomal and incomplete dominance of resistance. A direct test of a monogenic model and Land's method suggested that resistance to emamectin was controlled by more than one locus. CONCLUSION: Instability of resistance and lack of cross-resistance to other insecticides suggest that insecticides with different modes of action should be recommended to reduce emamectin selection pressure.

Resistance to selected organochlorin, organophosphate, carbamate and pyrethroid, in Spodoptera litura (Lepidoptera: Noctuidae) from Pakistan.

The toxicity of the most commonly used insecticides of organochlorine, organophosphate, pyrethroid, and carbamate groups were investigated against Spodoptera litura (F.) (Lepidoptera: Noctuidae) populations collected for three consecutive years (2004-2006). For a chlorocyclodiene and pyrethroids tested, the resistance ratios compared with Lab-PK were in the range of 10- to 92-fold for endosulfan, 5- to 111-fold for cypermethrin, 2- to 98-fold for deltamethrin, and 7- to 86-fold for beta-cyfluthrin. For organophosphates and carbamates, resistance ratios were in the range of 3- to 169-fold for profenofos, 18- to 421-fold for chlorpyrifos, 3- to 160-fold for quinalphos, 6- to 126-fold for phoxim, 7- to 463-fold for triazophos, and 10- to 389-fold for methomyl and 16- to 200-fold for thiodicarb. Resistance ratios were generally low to medium for deltamethrin and beta-cyfluthrin and high to very high for endosulfan, cypermethrin, profenofos, chlorpyrifos, quinalphos, phoxim, triazophos, methomyl, or thiodicarb. Pairwise comparisons of the log LC50 values of insecticides tested for all the populations showed correlations among several insecticides, suggesting a cross-resistance mechanism. Integration of timely judgment of pest problem, delimiting growing of alternate crops such as arum, rotation of insecticides with new chemicals, and insect growth regulators in relation to integrated pest management could help in manageable control of this important pest.

Cross-resistance and genetics of resistance to indoxacarb in Spodoptera litura (Lepidoptera: Noctuidae).

Bioassays (at generation G1) with a newly collected field population of Spodoptera litura (F.) (Lepidoptera: Noctuidae) from Multan, Pakistan, showed resistance ratios of 15, 23, 37, and 16 for indoxacarb, spinosad, abamectin, and emamectin, respectively, compared with a laboratory susceptible population, Lab-PK. At G1, the field population was selected with indoxacarb by using single pair crosses. For Indoxa-SEL, bioassay at G4 found that selection increased resistance ratio to 95 for indoxacarb compared with Lab-PK. Selection with indoxacarb significantly increased resistance to spinosad and emamectin; however, resistance to abamectin was observed to drop. A significant reduction in the resistance to indoxacarb was observed in Indoxa-SEL at G9, indicating unstable resistance to indoxacarb; however, it was stable for fipronil. Synergism tests with microsomal oxidase and esterase-specific inhibitors suggested that the indoxacarb resistance was associated with microsomal oxidase. Reciprocal genetic crosses between Indoxa-SEL and Lab-PK populations indicated that resistance was autosomal and incompletely dominant. Tests of monogenic inheritance suggested that resistance to indoxacarb was controlled by more than one locus.

Genetics and mechanism of resistance to deltamethrin in a field population of Spodoptera litura (Lepidoptera: Noctuidae).

BACKGROUND: Spodoptera litura (F.) causes enormous losses in many economically important crops. The genetics of insecticide resistance has been extensively studied in several insect pests, but there is a lack of information on S. litura. Therefore, the genetics and mechanisms of the resistance of S. litura to deltamethrin were investigated. RESULTS: Bioassays at generation G1 gave resistance ratios of 9, 5, 41, 52 and 49 for deltamethrin, cypermethrin, profenofos, chlorpyrifos and triazofos respectively, when compared with the susceptible Lab-PK strain. Bioassays at G4 with a deltamethrin-selected population (Delta-SEL) showed that selection gave resistance ratios of 63 and 7 for deltamethrin when compared with the Lab-PK and UNSEL strains respectively. Cross-resistance to other insecticides tested was observed in the selected population. A notable feature of the Delta-SEL strain was that resistance to deltamethrin, cypermethrin, profenofos and chlorpyrifos did not decline over the course of five generations. Synergism tests with microsomal oxidase (MO) and esterase-specific inhibitors indicated that the deltamethrin resistance was associated with MO and, possibly, esterase activity. Reciprocal crosses between the Delta-SEL and Lab-PK strains indicated that resistance was autosomal and incompletely dominant. A direct test of monogenic inheritance suggested that resistance to deltamethrin was controlled by more than one locus. CONCLUSION: Stability and dominance of resistance and cross-resistance suggest that insecticides with different modes of action should be recommended to reduce pyrethroid selection pressure.