{"id":2672,"date":"2019-02-15T17:54:53","date_gmt":"2019-02-15T08:54:53","guid":{"rendered":"http:\/\/163.180.4.222\/lab\/?p=2672"},"modified":"2019-02-15T17:54:53","modified_gmt":"2019-02-15T08:54:53","slug":"crispr-cas9-based-genome-editing-of-human-cells","status":"publish","type":"post","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=2672","title":{"rendered":"CRISPR-Cas9-Based Genome Editing of Human Cells"},"content":{"rendered":"

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CRISPR\/Cas9 systems are engineered versions of the Cas9 protein and guide RNA. \u00a0Typically, they are identical to the\u00a0Streptococcus pyogenes\u00a0<\/em>type II CRISPR systems, except that a single guide-RNA is used in place of the complementary crRNAs and tracrRNAs of the natural CRISPR system, and the Cas9 protein is codon-optimized for the cells intended to be transfected with the CRISPR\/Cas9 system\u00a0(Jinek et al. 2012)<\/a>. \u00a0For example, CRISPR\/Cas9 expression plasmids used to edit the genome of human cells have codons optimized for human cells, and thus are called humanized Cas9 (hCas9)\u00a0(Mali et al. Feb 2013)<\/a>.<\/p>\n

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\"Genome<\/a><\/p>\n

Figure 1: Natural vs. Engineered CRISPR systems. Natural CRISPR Pathway: 1. transcription of pre-crRNA and tracrRNA 2. binding of tracrRNA to pre-crRNA 3. cleavage of guide RNA from pre-crRNA 4. binding of inactive Cas9 nuclease to the guide RNA to produce the active Cas9 nuclease. Engineered CRISPR: 1. transcription of Guide RNA as a single sequence 2. transcription and translation of cas9 nuclease 3. binding of Guide RNA to Cas9 and Activation of Cas9 (original figure)<\/p>\n<\/div>\n

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As the guide RNA sequence determines the cut site of the Cas9 nuclease with high specificity, the re-targeting of the CRISPR\/Cas9 system to new sequences is far easier than re-targeting other engineered nucleases such as TALENs (transcription activator-like effector nuclease) or ZFNs (zinc finger nuclease). In fact, the cut site of a guide RNA-Cas9 nuclease complex can be reprogrammed to target just about any site that occurs immediately before a PAM sequence (5\u2032-NGG-3\u2032)\u00a0(Cong et al. 2013)<\/a>. \u00a0In addition, the efficiency of the nuclease is comparable or greater than that of TALENs or ZFNs, which is remarkable given the high specificity of the nuclease\u00a0(Mali et al. Feb 2013)<\/a>. \u00a0To understand how this high specificity and high efficiency are possible, check out our\u00a0mechanism<\/a>\u00a0page.<\/p>\n

\"Screen<\/p>\n

Figure 2: Cas9 cleaves DNA at a site specified by the protospacer adjacent motif (NGG) and the 20 nucleotide guide RNA complementary strand. (original figure)<\/p>\n<\/div>\n

Cas9, as a double-stranded DNA nuclease, can be used in conjunction with\u00a0Homology-Directed Repair<\/a>\u00a0to insert new genes or DNA sequences into a genome\u00a0(Mali et al. Feb 2013)<\/a>.<\/p>\n

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There are two distinct nuclease domains in Cas9 and therefore mutations in the active site of one nuclease can produce a\u00a0DNA nickase<\/a>\u00a0that produces single strand breaks\u00a0(Gasiunas et al. 2012)<\/a>.<\/p>\n

Mutations in the active sites of both nuclease domains results in a non-catalytic Cas9. \u00a0This protein-RNA complex can bind specific sequences of DNA and localize other effector molecules to that region of DNA, which can carry out\u00a0Transcriptional Control<\/a>\u00a0of the genome\u00a0(Jinek et al. 2012)<\/a>.<\/p>\n

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(\uc6d0\ubb38: \uc5ec\uae30<\/a>\ub97c \ud074\ub9ad\ud558\uc138\uc694~)<\/p>\n

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    CRISPR\/Cas9 systems are engineered versions of the Cas9 protein and guide RNA. \u00a0Typically, they are identical to the\u00a0Streptococcus pyogenes\u00a0type II CRISPR systems, except(more…)<\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":"","jetpack_publicize_message":"","jetpack_publicize_feature_enabled":true,"jetpack_social_post_already_shared":true,"jetpack_social_options":{"image_generator_settings":{"template":"highway","default_image_id":0,"font":"","enabled":false},"version":2},"jetpack_post_was_ever_published":false},"categories":[33,34,29],"tags":[],"class_list":["post-2672","post","type-post","status-publish","format-standard","hentry","category-do-biology","category-lets-do-chemistry","category-lets-do-science"],"aioseo_notices":[],"jetpack_publicize_connections":[],"jetpack_featured_media_url":"","jetpack-related-posts":[{"id":4118,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=4118","url_meta":{"origin":2672,"position":0},"title":"CRISPR-mediated live imaging of genome editing and transcription","author":"biochemistry","date":"September 23, 2019","format":false,"excerpt":"\u00a0 \u00a0 Tracking nucleic acids in living cells Fluorescence in situ hybridization (FISH) is a powerful molecular technique for detecting nucleic acids in cells. However, it requires cell fixation and denaturation. Wang\u00a0et al.\u00a0found that CRISPR-Cas9 protects guide RNAs from degradation in cells only when bound to target DNA. Taking advantage\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":4854,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=4854","url_meta":{"origin":2672,"position":1},"title":"Got mutation? \u2018Base editors\u2019 fix genomes one nucleotide at a time","author":"biochemistry","date":"November 19, 2019","format":false,"excerpt":"\u00a0 \u00a0 A new class of CRISPR-based tools efficiently corrects point mutations in cell lines, animal models and perhaps the clinic. \u00a0 \u00a0 Credit: Getty \u00a0 \u00a0 When Xingxu Huang began thinking about correcting disease-causing mutations in the human genome, his attention turned naturally to CRISPR\u2013Cas9. But it quickly became\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":2531,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=2531","url_meta":{"origin":2672,"position":2},"title":"CRISPR adapted to respond to infected cells","author":"biochemistry","date":"January 18, 2019","format":false,"excerpt":"\u00a0 \u00a0 By making a small change to the sequence of the Cas9 protein researchers can control the enzyme\u2019s activity. Credit: B. L. Oakes\u00a0et al.\/Cell \u00a0 \u00a0 \u00a0 Engineered tweaks to the popular gene-editing system allow it to fight viral infection. \u00a0 A bacterial enzyme that researchers often use to\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":1857,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=1857","url_meta":{"origin":2672,"position":3},"title":"CRISPR-Cas9 nuclease \uad00\ub828 \uba87 \uac00\uc9c0 \ub274\uc2a4","author":"biochemistry","date":"September 25, 2018","format":false,"excerpt":"\u00a0 \u00a0 CRISPR-Cas9\uacfc \uad00\ub828\ub41c \uba87 \uac00\uc9c0 \uc18c\uc2dd\uc785\ub2c8\ub2e4. (\uc6d0\ubb38: \uc5ec\uae30\ub97c \ud074\ub9ad\ud558\uc138\uc694~) \u00a0 CRISPR tool puts RNA on the record \u00a0 The bacterial-defence system CRISPR\u2013Cas can store DNA snippets that correspond to encountered viral RNA sequences. One such system has now been harnessed to record gene expression over time in bacteria. \u00a0\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":3361,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=3361","url_meta":{"origin":2672,"position":4},"title":"When genome editing goes off-target","author":"biochemistry","date":"April 19, 2019","format":false,"excerpt":"\u00a0 \u00a0 Editing DNA in eukaryotic cells with CRISPR-based systems has revolutionized the genome engineering field. Cas (CRISPR-associated) endonucleases are directed to a particular location in the genome by a short guide RNA, providing an easily programmable strategy to target any section of DNA. As of now, two CRISPR-based approaches\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":2540,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=2540","url_meta":{"origin":2672,"position":5},"title":"Precision CRISPR editing","author":"biochemistry","date":"January 18, 2019","format":false,"excerpt":"\u00a0 \u00a0 The most popular gene-editing tool, CRISPR-Cas9, generates breaks in the genome that are subsequently repaired by a mix of cellular pathways. Yet, the repair outcomes are not random. Using machine-learning algorithms to analyze large amounts of Cas9-mediated, genome-wide editing events in a range of cells, Shen\u00a0et al., Allen\u00a0et\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]}],"jetpack_sharing_enabled":false,"jetpack_shortlink":"https:\/\/wp.me\/p9Xo1j-H6","_links":{"self":[{"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/posts\/2672","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=2672"}],"version-history":[{"count":1,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/posts\/2672\/revisions"}],"predecessor-version":[{"id":2673,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/posts\/2672\/revisions\/2673"}],"wp:attachment":[{"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=2672"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=2672"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=2672"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}