{"id":54291,"date":"2020-08-26T19:54:00","date_gmt":"2020-08-26T16:54:00","guid":{"rendered":"https:\/\/en.buradabiliyorum.com\/barcoding-long-dna-quantifies-crispr-effects\/"},"modified":"2020-08-26T19:54:00","modified_gmt":"2020-08-26T16:54:00","slug":"barcoding-long-dna-quantifies-crispr-effects","status":"publish","type":"post","link":"https:\/\/buradabiliyorum.com\/en\/barcoding-long-dna-quantifies-crispr-effects\/","title":{"rendered":"#Barcoding long DNA quantifies CRISPR effects"},"content":{"rendered":"

“#Barcoding long DNA quantifies CRISPR effects<\/strong>”<\/p>\n

\n
\n
\n
\"Barcoding<\/img>
\n The\u00a0sequencing setup for the study: an Oxford Nanopore sequencer and a laptop computer. The screen in background shows the DNA strand fed through the sequencer. Credit: 2020 KAUST Mo Li.\u00a0
\n <\/figcaption><\/figure>\n<\/div>\n<\/div>\n

Current sequencing techniques lack the sensitivity to detect rare gene mutations in a pool of cells, which is particularly important, for example, in early cancer detection. Now, scientists at KAUST have developed an app<\/a>roach, called targeted individual DNA molecule sequencing (IDMseq), that can accurately detect a single mutation in a pool of 10,000 cells.<\/p>\n

\n <\/section>\n

Importantly, the team successfully used IDMseq to determine the number and frequency of mutations caused by the gene editing tool, CRISPR\/Cas9, in human embryonic stem cells. Clinical trials are underway to test CRISPR’s safety to treat some genetic diseases. “Our study revealed potential risks associated with CRISPR\/Cas9 editing and provides tools to better study genome editing outcomes,” says KAUST bioscientist Mo Li, who led the study.<\/p>\n

IDMseq is a sequencing technique that involves attaching a unique barcode to every DNA molecule in a sample of cells and then making a large number of copies of each molecule using a polymerase chain reaction (PCR). Copied molecules carry the same barcode as the original ones.
\nA bioinformatics tool kit, called variant analysis with unique molecular identifier for long-read technology<\/a> (VAULT), then decodes the barcodes and places similar molecules into their own “bins”, with every bin representing one of the original DNA molecules. VAULT uses a combination of algorithms to detect mutations in the bins. The process works especially well with third-generation long-read sequencing technologies and helps scientists detect and determine the frequency of all types of mutations, from changes in single DNA letters to large deletions and insertions in the original DNA molecules.
\nThe approach successfully detected a deliberately caused gene mutation that was mixed with a group of wild-type cells at ratios of 1:100, 1:1,000 and 1:10,000. It also correctly reported its frequency.
\nThe researchers also used IDMseq to look for mutations caused by CRISPR\/Cas9 genome editing. “Several recent studies have reported that Cas9 introduces unexpected, large DNA deletions around the edited genes, leading to safety concerns. These deletions are difficult to detect and quantitate using current DNA sequencing strategies. But our approach, in combination with various sequencing platforms, can analyze these large DNA mutations with high accuracy and sensitivity,” says Ph.D. student Chongwei Bi.
\nThe tests found that large deletions accounted for 2.8-5.4 percent of Cas9 editing outcomes. They also discovered a three-fold rise in single-base DNA variants in the edited region. “This shows that there is a lot that we need to learn about CRISPR\/Cas9 before it can be safely used in the clinic,” says Yanyi Huang of Peking University, who is an international collaborator co-funded by KAUST.
\nIDMseq can currently sequence only one DNA strand, but work to enable double-strand sequencing could further improve performance, say the researchers.<\/p>\n

\n<\/hr>\n
\n<\/hr>\n
\n

More information:<\/strong>
\n Chongwei Bi et al, Long-read individual-molecule sequencing reveals CRISPR-induced genetic heterogeneity in human ESCs, Genome Biology<\/i> (2020). DOI: 10.1186\/s13059-020-02143-8\n <\/div>\n

\n Citation<\/strong>:
\n Barcoding long DNA quantifies CRISPR effects (2020, August 26)
\n retrieved 26 August 2020
\n from https:\/\/phys.org\/news<\/a>\/2020-08-barcoding-dna-quantifies-crispr-effects.html<\/p>\n

This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no
\n part may be reproduced without the written permission. The content is provided for information purposes only.<\/p><\/div>\n<\/p><\/div>\n

\n

If you want to read more Like this articles, you can visit our Science category.<\/a><\/span><\/strong>\n<\/p><\/blockquote>\n

\n

if you want to watch Movies<\/a> or Tv Shows go to Dizi.BuradaBiliyorum.Com<\/a> <\/span> for forums sites go to Forum.BuradaBiliyorum.Com<\/a><\/span><\/strong><\/p>\n<\/blockquote>\n","protected":false},"excerpt":{"rendered":"

“#Barcoding long DNA quantifies CRISPR effects” The\u00a0sequencing setup for the study: an Oxford Nanopore sequencer and a laptop computer. The screen in background shows the DNA strand fed through the sequencer. Credit: 2020 KAUST Mo Li.\u00a0 Current sequencing techniques lack the sensitivity to detect rare gene mutations in a pool of cells, which is particularly…<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"fifu_image_url":"","fifu_image_alt":"","footnotes":""},"categories":[16],"tags":[59103,10663],"class_list":["post-54291","post","type-post","status-publish","format-standard","hentry","category-sciencee","tag-barcoding-long-dna-quantifies-crispr-effects","tag-biotechnology-molecular-computational-biology"],"_links":{"self":[{"href":"https:\/\/buradabiliyorum.com\/en\/wp-json\/wp\/v2\/posts\/54291","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/buradabiliyorum.com\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/buradabiliyorum.com\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/buradabiliyorum.com\/en\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/buradabiliyorum.com\/en\/wp-json\/wp\/v2\/comments?post=54291"}],"version-history":[{"count":0,"href":"https:\/\/buradabiliyorum.com\/en\/wp-json\/wp\/v2\/posts\/54291\/revisions"}],"wp:attachment":[{"href":"https:\/\/buradabiliyorum.com\/en\/wp-json\/wp\/v2\/media?parent=54291"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/buradabiliyorum.com\/en\/wp-json\/wp\/v2\/categories?post=54291"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/buradabiliyorum.com\/en\/wp-json\/wp\/v2\/tags?post=54291"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}