Several important techniques for inventorying whole genome detection of CRISPR off-target sites
Release date: 2017-05-10 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. Technique based on integrase-deficient lentiviral vector (IDLV) This technology can be used for both CRISPR and TALENs: in the DNA repair process of non-homologous end joining (NHEJ), the linear double-stranded IDLV genome is preferentially integrated into double-strand breaks (DSB). According to previously published articles, these IDLVs were able to introduce ZFN-treated cells to label the location of the DSB. Later, using linear amplification-mediated PCR, the integration site of IDLV can be located to reveal whether IFN hits the target site. Inspired by this approach, researchers at the Beckman Institute in the City of Hope and the First Affiliated Hospital of Zhejiang University applied it to the CRISPR and TALENs. The researchers transfected HEK cells with plasmids expressing CRISPR/Cas9 nuclease or TALENs, and then transduced IDLV. The gene carried by this IDLV confers resistance to antibiotics and puromycin. After nuclease formation of DSB, IDLV integration is allowed, increasing the number of clones resistant to puromycin two to three times. For CRISPR/Cas9 and TALEN-treated cells, the researchers identified clustered IDLV integration sites (CLIS) within 60 bp of the target cleavage site. When they looked for CLIS other than the WAS and TAT genes, no off-target sites were found in TALEN-treated cells, and some were found in CRISPR/Cas9 nucleases. They then used chip prediction and deep sequencing to determine the sensitivity of the IDLV analysis and found it to detect off-target cuts as low as 1%. BLESS Scientists have proposed a new method for DNA double-strand breaks (DSBs) mapping using genome-wide techniques. This mapping is based on single nucleotides and is called BLESS (direct in situ breaks labeling, enrichment on streptavidin and Next-generation sequencing). The researchers used the human and mouse cells and different DNA double-strand breaks (DSBs) inducing factors and sequencing platforms to validate the BLESS method. BLESS recognizes telomere ends, SCE endonuclease-induced DNA double-strand breaks, and complex DSB genomes. As a proof of principle, we characterized human replication by a sensitive genome and identified > 2,000 non-uniformly distributed amphidimycin-sensitive regions (ASRS), which are over-enriched and rich in microsatellite repeats. . Human cancer rearrangements are also rich in ASRS, and many cancer-related genes are extremely sensitive to replication stress. The researchers say the new method is suitable for genome-wide mapping of DSBs under various nuclear assay conditions, and its specificity and resolution are not achievable with current technology. SITE-Seq Researchers have developed a new biochemical method to selectively enrich and identify the end of the marker genomic DNA by sequencing (SITE-Seq method) to find the cleavage site of Cas9 in purified genomic DNA. SITE-Seq is a biochemical assay that allows genomic DNA to be digested from a range of sgRNP concentrations, from lowest to highest, allowing for high-resolution and low-cut sensitivity off-target identification, so researchers can meticulously Comprehensive examination of possible off-target sites in cells, detection of mutation frequency and functional cell effects. Moreover, SITE-Seq can also create highly sequenced libraries of sgRNP-cleaved fragments, helping to perform specific analysis with minimal read depth, a critical feature of SITE-Seq as a high-throughput guide selection tool. Finally, the researchers used SITE-Seq to map a set of sgRNP biochemical cleavage maps that examined the Cas9 specificity of sgRNAs targeting the human genome, the number of which depends on the sgRNA sequence and nuclease concentration, at lower concentrations. The sites identified below showed a higher propensity for off-target mutations in the cells, and the list of off-target sites also indicated that intracellular Cas9 activity was affected by sgRNP delivery, cell type and duration of exposure to nucleases. Nature Methods: CRISPR-Cas9 Whole Genome Shear Site Map CIRCLE-seq Researchers at Massachusetts General Hospital and Harvard University introduced a new in vitro screening method, CIRCLE-seq. They believe that this method performs better than existing cell or biochemical methods in identifying genome-wide CRISPR/Cas9 off-target mutations. The researchers believe that this method can identify off-target mutations associated with cell type-specific single nucleotide polymorphisms, proving that it is expected to generate a personalized specificity map. At the same time, CIRCLE-seq provides a fast and comprehensive method for identifying genome-wide off-target mutations. The researchers also tested the sensitivity of the new method. They obtained off-target results for the guide RNA against the HBB gene and compared this map to the map obtained by another method of identification (Digenome-seq). They found that CIRCLE-seq identified 26 of the 29 off-target sites found in Digenome-seq and also found 156 new sites. A new strategy for comprehensive detection of CRISPR off-target effects Source: Biopass Medical Mask,N95 Medical Mask,Black Medical Mask,Kn95 Medical Mask Zhejiang Lanhine Medical Products Ltd. , https://www.lanheyiliao.com