Release date: 2015-01-06
When people refer to so-called enabling technologies, inventions like printers, or the discovery of anesthetics, will come to our minds. For scientists, the CRISPR-Cas9 system is such a system.
This bacterial immune system is resistant to viruses by integrating short repeats in viral DNA into the bacterial genome. When a bacterium (or its progeny) infects a virus for the second time, these repeats can target the invading complementary DNA through a nuclease and destroy it.
In 2013, several research groups used this system to edit eukaryotic genes. Later, in many applications, CRISPRs rose rapidly. Many different species realized gene deletion and insertion in the genome, and activated or inhibited gene transcription. But as this system becomes more popular, there are more and more questions about its specificity and effectiveness.
How to improve the specificity of the targeting RNA (gRNA), which can lead Cas9 nucleases to the target of the genome, Nature Biotechnology recommends cutting off gRNAs (Nature subsurgence: problem-solving off-target problem), reducing off-target The problem, and does not affect the target activity, while another study group recommends longer-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.
This is obviously a somewhat contradictory suggestion. The former researchers pointed out that the length of the gRNA target region was simply truncated. It was found that the off-target mutations were significantly reduced in the previously detected off-target sites, compared with the full-length gRNA. The mutation frequency of some sites is even reduced by more than 5,000 times. It is important that these shortened gRNAs (called tru-gRNAs) are as effective as full-length gRNAs when targeting their intended target DNA. The latter found that selecting the appropriate target sequence and optimizing gRNA and Cas9 could avoid or reduce the occurrence of off-target mutations.
Another question is how to screen for off-target and how much impact it has on the study. Methods for verifying mismatched candidate sites for gRNAs may miss some of the key cleavage sites, and whole-genome sequencing is not very effective. Currently we need a sensitive and unbiased approach to label Cas9 target sites, and Let them be identified throughout the genome.
Other methods that make this system more specific are the replacement of Cas9 nucleases with paired replacement nickases (the former can only cleave one strand of DNA at a time), or the fusion of Cas9 mutations into Fok1. Activated nucleases. In addition, Cas9 from different species can also increase the flexibility to target multiple sites, further improving the delivery system of the Cas9-gRNA complex and helping to increase efficiency.
Although Cas9 has unlimited potential, it is also a bacterial protein. For some applications, especially for clinical applications, it is also necessary to find eukaryotic nucleases for replacement. For plants, some Argonaute nucleases can target DNA through small RNAs, which is a direction that genome editors can consider.
Source: Biopass
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