Cell survival rate increased by 7 times! More accurate CRISPR was born
May 10, 2018 Source: WuXi PharmaTech
Window._bd_share_config={ "common":{ "bdSnsKey":{ },"bdText":"","bdMini":"2","bdMiniList":false,"bdPic":"","bdStyle":" 0","bdSize":"16"},"share":{ }};with(document)0[(getElementsByTagName('head')[0]||body).appendChild(createElement('script')) .src='http://bdimg.share.baidu.com/static/api/js/share.js?v=89860593.js?cdnversion='+~(-new Date()/36e5)];Editing DNA using CRISPR gene editing techniques is often referred to as scraping a press release with scissors. However, it is relatively easy to clip a word, delete a single character or immediately understand the effect of the clip on the meaning of the text. Scientists hope to use CRISPR technology to remove the genetic mutations in the DNA and replace them with normal DNA sequences. To achieve this goal, the accuracy of the CRISPR technology still needs to be improved.
▲CRISPR technology is often referred to as “genetic scissors†(Source: 123RF.com)
Researchers at the Joint Biometrics Initiative (JIMB), a joint effort between Stanford University and the National Institute of Standards and Technology (NIST), have developed a next-generation CRISPR technology platform called MAGESTIC. This platform transforms CRISPR technology from a crude scrapbooking tool to a sophisticated word processor that uses precise "query" and "replace" functions to accurately edit genetic material. This achievement was published in Nature Biotechnology.
Editing the genome using CRISPR technology requires the following steps: 1. Cutting at a specific site in the cellular DNA; 2. Accurately introducing the sequence to be added to the genome at the site of the incision; 3. Accurately observing the gene The consequences of the editor. Current CRISPR technology uses guide RNA (gRNA) to direct precise cleavage of DNA sequences, and donor DNA helps introduce DNA sequences that need to be added to specific sites into the genome.
Currently, gRNA can guide precise cleavage of specific sites in the genome, but the next step of DNA repair is often not precise enough. Many times the repair process introduces random mutations, and artificially expressed donor DNA sequences cannot be efficiently integrated into the genome. This causes a large number of cells to die during the gene editing process. For cells, it is a challenging task to have DNA repair mechanisms find suitable donor DNA in DNA sequences containing millions to hundreds of millions of base pairs. An advancement in this area of ​​the MAGESTIC platform is the use of a method called "active donor recruitment" to recruit synthetic donor DNA fragments around the cleavage site. This recruitment method has increased cell survival by a factor of seven!
â–²MAGESTIC platform uses cell barcodes in new ways, adding a new level of precision to CRISPR gene editing (Source: Kelly Irvine/NIST)
Another significant difference between MAGESTIC and previous high-throughput CRISPR gene editing technologies is the design of cell barcodes. In the past, researchers used plasmids to express gRNAs and stored barcode sequences to track cell mutations that replicated as cells grow and divide and are passed on to the next generation of cells. However, the amount of plasmid DNA in each cell can vary from 10 to 40, which can result in the number of barcodes not being used to accurately detect the number of cells. The new MAGESTIC technology platform integrates barcodes into the chromosomes of cells, an improvement that stabilizes the number of barcodes, allowing them to predict the number of cells more accurately.
Although there has been significant progress in DNA sequencing and editing techniques over the past 20 years, scientists still lack understanding of the functions of natural variability in the genome and their impact on disease. MAGESTIC enhances understanding of natural variation by precisely editing each naturally occurring genetic variation and comparing it to other genetic variations. Another advantage of MAGESTIC is the ability to perform high-throughput gene editing on millions of cells in a single tube, while past technology platforms required different experiments to edit each gene variant.
"The current technological advancement allows us to not only sequence every base pair in the genome, but also to modify them. But we still need to better understand the consequences of the modification," said Stanford University, senior author of the paper. Dr. Lars Steinmetz, a professor of genetics, said: "The MAGESTIC platform is like letting us modify a single character in the genome book and then observe the impact of this modification on the meaning of the text. And our donor recruitment method allows new information to be added. It is placed exactly on the page where the cut appears."
Reference materials:
[1] Taking CRISPR from clipping scissors to word processor
[2] Multiplexed precision genome editing with trackable genomic barcodes in yeast
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