In 2013, researchers from the Massachusetts General Hospital found an important limitation of using CRISPR-Cas RNA-directed nucleases: they generate redundant DNA mutations at sites other than the intended target. Since then, studies have directly demonstrated the serious off-targetness of CRISPR/Cas9, that is, the technology can cause non-specific cleavage, causing mutations in non-targeting sites of the genome, which will lead to uncertainty in research results and a large increase in research work. This problem severely limits the application of Cas9. Therefore, scientists hope to detect off-target shear by genome-wide detection. In recent years, many experimental methods for detecting off-target shear enzyme activity have been developed. For example, two technologies recently reported by Nature Methods, what are these technologies, and what are the advantages. Let's see: GUIDE-seq In the past, when detecting CRISPR-Cas nuclease-induced off-target DNA cleavage, it was often assumed that the off-target site was similar to the target site. GUIDE-seq is the first method that doesn't need to be done, and it's quite sensitive. A group of researchers used a short double-stranded oligonucleotide to tag CRISPR-Cas-induced off-target cleavage and sequence the genomic region in which these tags are located to determine the location of the off-target mutation. Studies have shown that even if the frequency of occurrence of an off-target mutation is as low as 0.1%, GUIDE-seq can be detected. Since many off-target mutations occur where the target site is very different, the number and location of off-target DSBs is difficult to predict. Existing tools primarily predict off-target mutations by analyzing target sequences, and the researchers compared these tools to GUIDE-seq. Studies have shown that the above tools are much worse than GUIDE-seq in predicting validated off-target sites and incorrectly detect sites that are not actually digested. In addition, the researchers compared GUIDE-seq and ChIP-seq (detection of protein-DNA binding) and demonstrated that ChIP-seq is not a reliable method for identifying CRISPR-Cas off-target DSB. Digenome-seq Researchers from Seoul National University and Seoul Institute of Basic Sciences published a study in Nature's subsidiary Nature Methods, which successfully confirmed that CRISPR-Cas9 has precise targeting in human cells, and they have developed a powerful A sensitive, unbiased and cost-effective method, Digenome-seq, detects CRISPR/Cas9 off-target effects in human cells across the genome. In this study, the researchers said that although RNA-directed genome editing through the CRISPR-Cas9 system has been widely used in biomedical research, the genome-wide targeting specificity of Cas9 nucleases remains controversial. To this end, they proposed a method Digenome-seq that uses genome sequencing to find targeted and off-target sequences generated by possible mutations in CRISPR-Cas9. They used Cas9 nuclease to digest human genomic DNA in vitro and then performed whole-genome sequencing. This in vitro digestion produces a unique pattern of targeting and off-target sequences that can be determined by calculation. In addition, the addition of a guanine nucleotide that constitutes CRISPR-Cas9 at the end of the sgRNA, the researchers successfully prepared this highly developed programmable nuclease, which has no measurable off-target effect in the human genome. Since then, the research team has released an upgraded version of Digenome-seq, and they have used this technology to simultaneously analyze the specificity of 11 CRISPR-Cas9 nucleases in the entire genome, saving significant time and time required for CRISPR off-target analysis. Funding. The researchers first extracted the genomic DNA from human cells, then digested with multiple sgRNA-Cas9 combinations, and finally performed genome-wide sequencing. Using a new DNA-cleaving scoring system, they identified the cleavage pattern of Cas9 in the genome, the nature of the target and off-target sites. Studies have shown that sgRNAs transcribed from double-stranded oligonucleotides cause many off-target events, whereas sgRNAs transcribed from plasmid templates do not have such problems. Multipleized Digenome-seq captures many benign off-target sites that are missed by other methods. The researchers also gave guidance on reducing the off-target effect of CRISPR-Cas9. Other Specific Function Centrifuge Other Specific Function Centrifuge,Pcr Plate Centrifuge,Specific Function Centrifuge,Medical Refrigerated Centrifuge Guangdong Widinlsa International Co.Ltd , https://www.gdwidinlsa.com