![]() ![]() There are three main types of CRISPR-based genome perturbation technologies that researchers can use for pooled screens (Fig. By transducing the sgRNA lentivirus pool into desired cell types (often at a multiplicity of infection much smaller than one to ensure less than one viral particle per cell), high-throughput, parallel loss-of-function genomic perturbations can be carried out to infer their functional relevance. The oligo library can be cloned onto a lentiviral vector system to facilitate cellular delivery, integration, and expression. High-throughput DNA synthesis platforms can generate a library of oligos with various sequence features (hundred of thousands or even millions), with each oligo encoding a different sgRNA sequence, and thus a different DNA target. The intriguing programmable RNA-guided DNA targeting feature of CRISPR-Cas allows it to be scaled up to target many genomic sites in parallel in one experiment. ![]() Fusing dCas molecules to transcriptional or epigenetic effector domains allows sequence-specific gene regulation for either gene activation (CRISPRa) or repression (CRISPRi). In addition to editing, nuclease-deactivated Cas (dCas) molecules have been engineered by introducing silencing mutations to abrogate the nuclease activity. This cutting mechanism on the genomic DNA triggers host non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways, which typically leads to loss-of-gene function. Taking the most commonly used CRISPR-Cas9 system as an example, the Streptococcus pyogenes Cas9 protein can complex with a 110-nucleotide (nt) sgRNA containing a 20-nt sequence that complementarily binds to the target DNA region and induces a double-stranded break (DSB). Due to its programmable nature using customizable single-guide RNAs (sgRNAs), CRISPR-Cas has enabled powerful pooled screens to explore the function of genetic perturbations at a genome-wide scale. where the agencies (or power companies) have installed gauges to monitor water flows and levels.Clustered regularly interspaced palindromic repeats (CRISPR) and the CRISPR-associated (Cas) proteins are a class of bacteria-encoded, RNA-guided, programmable DNA targeting and cleavage systems. Examples of this situation are the large reservoirs in the Rockies and Western U.S. Some river or dam/pool water level gauges will be referred to using feet (or meters) above mean sea level (MSL). This bottom limit/reading of these gauges is usually due to the position of the gauge in or along the river. In contrast, a few river gauges will not read below a certain level (which may be above it's respective gauge zero). When they do go below zero, it is usually a sign of a prolonged dry spell. Not many sites in the region actually go below zero ( Renovo and Williamsport are two examples that do). There is a collaboration between many government agencies in order to settle on a gauge zero, and which Datum (way of mapping the earth) will be used as a basis. If the level was to change up or down just to avoid having a negative value, it might become confusing for them to remember the new level which would impact their home. They know that the water reaches the front steps of their house at 17 feet (using the current values). If the gauge zero level was changed on a whim, all the historical data for the gauge site could be rendered useless or at least very confusing.įor example, it may be difficult to explain to people who have been used to having a certain river level/guage reading as a personal threshold. Still, the gauge zero datum levels are not changed to keep continuity.įor example, most of the gauges in the Susquehanna Basin experienced their record Flooding during Agnes in 1972. Silt may deposit in the river channel over time (filling the channel up), or the channel bottom may be scoured out to a deeper level by strong currents. ![]() These gauge zero levels are not changed very often. ![]() That gauge zero level is chosen considering many factors, like the USGS references (or benchmarks) that are near to the gauge site, or an historical level that may have been used for a hundred years or more. It means that the gauge is reading at or below the agreed-upon zero level. In summary, when a river gauge reads zero or in the negative numbers - it does not mean that the river has gone totally dry or is running below ground. A river's stage at a point (a gauge reading) is not an absolute measure of the depth of the water in the channel, rather it is a depth with respect to an historical Datum level. ![]()
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