En distintas especies vegetales, mutaciones específicas en el gen de la acetolactato sintasa (ALS) conducen a sustituciones de aminoácidos que confieren resistencia a herbicidas. La sustitución ...espontánea de Pro197 por His en el gen ALS de L. serriola (lechuga salvaje) generó resistencia a sulfonilureas e imidazolinonas que por cruzamiento pudo transferirse a lechuga cultivada (L. sativa). Nuestro objetivo es establecer la edición génica en lechuga por edición de base, modificando el codón Pro197 del gen ALS (LsALS) con cambios C por T que lo sustituyen por Ser197 o Leu197. Este trabajo describe la selección y armado del vector de edición, y su delivery por transformación genética. Mediante amplificación por PCR se logró identificar la región target de LsALS y se comprobó la ausencia de variantes alélicas. Se evaluó el número de potenciales off targets por análisis in sílico. Para la construcción del vector de edición se partió del plásmido pXSE901BG (Addgene), confirmando su identidad por antibiograma, PCR y análisis de perfiles de restricción. En el vector se reemplazó el cassette de resistencia a fosfinotricina por otro de resistencia a kanamicina y se incorporó la secuencia espaciadora del gRNA. Por PCR y secuenciación se confirmó la inserción de una única secuencia espaciadora en la posición y dirección correcta. Por último, se trasformó establemente la var. Grand Rapids de lechuga. Se obtuvieron 8 eventos diferentes, con una eficiencia del 6%, similar a la lograda previamente con otros vectores.
As CRISPR-based editing moves into therapeutic and clinical applications, it is critical to screen, select, and confirm activity of CRISPR guide RNAs (gRNA). This includes a feature to minimize ...unintentional genomic alterations from factors like manufacturing errors, cross-contamination, and other genotoxic effects. Leveraging experience in gRNA synthesis and manufacturing, we have done substantial work to ensure efficient on-target editing while reducing unintended events and toxicity.
We have developed a method to characterize genome editing levels below 0.1% that are potentially attributable to low levels of cross contamination during manufacturing events. At the outset, our custom arrayed synthetic guide RNA libraries allow for accelerated guide screening and selection. Coupled with our chemically modified gRNAs, which include 2’-O-methyl and 2’-fluoro RNA nucleotides, gRNA libraries are an efficient way to execute CRISPR knockout screens. We enable a wide selection of guide formats and purifications, including HPLC-purified guides. Scale-up synthesis with HPLC-purified guides provide a stage-specific improvement over our current RUO sgRNAs, which provide improved on target editing with a reduction in truncated products of synthesis. To support research and developmental stage-specific needs, we offer large scale RUO gRNAs, engineering run gRNAs, as well as cGMP gRNAs manufactured in an ICH Q7 compliant facility. Our workflows and services include solutions for all CRISPR applications, with support, expertise, and documentation to meet the needs of the genome editing community pursuing therapeutic applications.
Recent experimental efforts of CRISPR-Cas9 systems have shown that off-target binding and cleavage are a concern for the system and that this is highly dependent on the selected guide RNA (gRNA) ...design. Computational predictions of off-targets have been proposed as an attractive and more feasible alternative to tedious experimental efforts. However, accurate scoring of the high number of putative off-targets plays a key role for the success of computational off-targeting assessment.
We present an approximate binding energy model for the Cas9-gRNA-DNA complex, which systematically combines the energy parameters obtained for RNA-RNA, DNA-DNA, and RNA-DNA duplexes. Based on this model, two novel off-target assessment methods for gRNA selection in CRISPR-Cas9 applications are introduced: CRISPRoff to assign confidence scores to predicted off-targets and CRISPRspec to measure the specificity of the gRNA. We benchmark the methods against current state-of-the-art methods and show that both are in better agreement with experimental results. Furthermore, we show significant evidence supporting the inverse relationship between the on-target cleavage efficiency and specificity of the system, in which introduced binding energies are key components.
The impact of the binding energies provides a direction for further studies of off-targeting mechanisms. The performance of CRISPRoff and CRISPRspec enables more accurate off-target evaluation for gRNA selections, prior to any CRISPR-Cas9 genome-editing application. For given gRNA sequences or all potential gRNAs in a given target region, CRISPRoff-based off-target predictions and CRISPRspec-based specificity evaluations can be carried out through our webserver at https://rth.dk/resources/crispr/ .
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•HIV-1 Gag protein adopts a compact (C-Gag) form in cells.•C-Gag formation requires both MA and NC RNA-binding domains.•C-Gag is stabilized by an intramolecular interaction between MA ...and CA domains.•Mutation altering C-Gag formation reduce infectious pseudoparticles production and infectivity.
HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm. At the plasma membrane, the membrane competes with gRNA for Gag binding, resulting in a transition to the extended form of Gag found in immature particles with MA bound to membrane lipids and NC to gRNA. The Gag compact form was previously evidenced in vitro. Here, we demonstrated the compact form of Gag in cells by confocal microscopy, using a bimolecular fluorescence complementation approach with a split-GFP bipartite system. Using wild-type Gag and Gag mutants, we showed that the compact form is highly dependent on the binding of MA and NC domains to RNA, as well as on interactions between MA and CA domains. In contrast, Gag multimerization appears to be less critical for the accumulation of the compact form. Finally, mutations altering the formation of Gag compact form led to a strong reduction in viral particle production and infectivity, revealing its key role in the production of infectious viral particles.
A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a ...target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work in Drosophila and mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs in Drosophila melanogaster targeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.
La implementación de las nuevas tecnologías Next- Generation Sequencing (NGS), plantea nuevos retos. Entre ellos, distinguir las variantes genéticas con impacto o significancia clínica para los ...pacientes. Dichas variantes no siempre son mutaciones del tipo nonsense o missense, y se les denomina variantes de significado incierto (Variants of Uncertain Significance, VUS). En un estudio previo, nuestro centro de investigación (CIGBM) identificó seis nuevas VUS en el gen BRCA1 en 18 familias peruanas. De ellas, la variante intrónica c.5074+28T>A fue encontrada en siete familias con cáncer hereditario de mama y ovario; y puede ser considerada como potencialmente patogénica, según análisis in silico de eventos de empalme (ESEfinder). El objetivo del estudio fue modelar, con la técnica CRISPR/Cas9, la VUS c.5074+28T>A en líneas celulares de tejido mamario sano (SVCT) y de cáncer de mama (MDA-MB-231). Los cultivos celulares libres de Mycoplasma fueron transfectados por electroporación con los complejos ribonucleoproteicos, Cas9 y gRNA. Se han generado clones genéticamente modificados que están siendo caracterizados por secuenciamiento Sanger en la región de interés y otras para descartar efectos offtarget. Asimismo, se evaluarán los efectos de esta variante en los sitios de empalme del ARN mediante ensayos de RNA-splicing. Una vez modelada, el efecto de esta variante podrá ser evaluado a través de estudios funcionales. Los resultados obtenidos sirven como data preliminar para desarrollar una plataforma de modelamiento con CRISPR/Cas9 y el estudio de VUS en pacientes peruanas con cáncer de mama.
Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, ...Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ.
In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined "3n"-, "3n + 1"-, or "3n + 2"-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to "3n + 1"-bp, "3n + 2"-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions.
NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.
The most precise, effective, and widely used tool for editing the genome is currently the clustered regularly interspaced short palindromic repeat (CRISPR), which represents the prokaryotes adaptive ...immune defense. The CRISPR/Cas-9 genome editing system relies on two key elements; mainly the guide RNA (gRNA) and CRISPR-associated (Cas-9) proteins. A complementary base pair in the designed sgRNA allows it to recognize the target sequence in the gene of interest. Either the non-homologous end joining or the homology-directed repair can be used to repair the double-stranded breaks, which are created by Cas-9 nuclease at a position upstream from a protospacer adjacent motif. The modified genome-editing tool CRISPR/Cas-9 has numerous applications in the various fields, such as biotechnology and medicine. Moreover, it is being studied for cancer management; Human Immunodeficiency virus (HIV), and as a gene therapy for several genetic diseases, including cystic fibrosis; sickle cell disease, and Duchenne muscular dystrophy. However, immunogenicity; off-target effect, and efficient delivery systems withstand against its spread in the clinical applications until introducing an improvement. The aim of this review was to summarize how the various CRISPR systems work; their important medical applications, and their limitations.
CRISPR/Cas9 is the genome-editing technology that is most widely used around the world. Its widespread adoption is largely due to its simplicity and ease of use. Here, we introduce the construction ...of vectors and genome editing of the target gene in cells using the CRISPR/Cas9 system.
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