{"id":752,"date":"2023-08-10T13:16:30","date_gmt":"2023-08-10T20:16:30","guid":{"rendered":"https:\/\/dornsife.usc.edu\/pzqin-lab\/?page_id=752"},"modified":"2023-08-10T13:16:31","modified_gmt":"2023-08-10T20:16:31","slug":"dna-target-discrimination-by-crispr","status":"publish","type":"page","link":"https:\/\/dornsife.usc.edu\/pzqin-lab\/dna-target-discrimination-by-crispr\/","title":{"rendered":"DNA Target Discrimination by CRISPR"},"content":{"rendered":"\n\n \n \n\n\n\n\n\n\n\n
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CRISPR-Cas [Clustered-Regularly-Interspaced-Short-Palindromic-Repeats (CRISPR) and CRISPR-associated (Cas) proteins] systems has ushered in a revolution in genome engineering and manipulation that is still rapidly evolving. Among the large CRISPR family, CRISPR-Cas9 and Cas12a are RNA-guided nucleases that natively cleave double-stranded DNAs at specific sites. The power of Cas9 and Cas12a lies in the programmability afforded by tunning the RNA guide sequence to form Watson-Crick pairs with a desired DNA site. However, this also subjects these enzymes to undesired off-target effects, in which erroneous binding and\/or cleavage occurs on off-target genomic sequences containing imperfect RNA\/DNA complementarity. Extensive mechanistic investigations have revealed that Cas9 and Cas12a interrogate a DNA target via a series of coordinated conformational changes. Further in-depth understanding on the structure-dynamics-function relationship that governs their target selection is the key to combat the off-target effect, and remains an area under active investigation.<\/p>\n

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Cas9 and Cas12a Interrogate DNA Targets via Coordinated Conformational Changes<\/strong><\/em><\/h5>\n

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The Qin lab studies Cas9 and Cas12a mechanisms by probing structure and dynamics of on-target and off-target complexes using a combination of spin-labeling, fluorescence spectroscopy, and other biophysical methods, and correlating the finding to functional outcomes obtained with detailed enzyme kinetics analysis, bioinformatics, and other biochemical approaches. Work has uncovered key specificity check-points built upon equilibrium in DNA unwinding, and shed light on the similarity and difference between Cas9 and Cas12a. Studies are on-going on further delineating the connection between sequence-dependence DNA duplex features (DNA shape) and Cas9a\/Cas12a target selection, and incorporating the finding in developing gene editing applications with enhanced specificity.<\/p>\n

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Representative Work on Cas9<\/strong><\/span><\/em><\/h3>\n\n\n\n\n\n\n\n\n
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Spin-Labeling Detects Cas9-Induced DNA Unwinding<\/a><\/p>\n<\/td>\n

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Parallel-Sequential Model for Analyzing Cas9 Cleavage<\/a><\/p>\n<\/td>\n<\/tr>\n

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PAM-Distal Partial DNA Unwinding with Truncated Guides<\/a><\/p>\n<\/td>\n

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Intrinsic DNA Duplex Stability Tunes Cas9 Activity with Truncated Guides<\/a><\/p>\n<\/td>\n<\/tr>\n

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Spin-Labeling Reveals Cas9 Domain Motion During Activation<\/a><\/p>\n<\/td>\n

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2Pro Variants with Enhanced Specificity<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Representative Work on Cas12a<\/strong><\/em><\/span><\/h3>\n\n\n\n\n
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Cas12a Binds Flexible DNA Duplexes without RNA-DNA Complementarity<\/a><\/p>\n<\/td>\n

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A DNA Unwinding Equilibrium Serves as a Checkpoint for Cas12a Target Discrimination<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n<\/div>\n\n\n <\/div><\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":545,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"class_list":["post-752","page","type-page","status-publish","hentry"],"acf":[],"yoast_head":"\nDNA Target Discrimination by CRISPR - Peter Qin Lab<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/dornsife.usc.edu\/pzqin-lab\/dna-target-discrimination-by-crispr\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"DNA Target Discrimination by CRISPR - Peter Qin Lab\" \/>\n<meta property=\"og:url\" content=\"https:\/\/dornsife.usc.edu\/pzqin-lab\/dna-target-discrimination-by-crispr\/\" \/>\n<meta property=\"og:site_name\" content=\"Peter Qin Lab\" \/>\n<meta property=\"article:modified_time\" content=\"2023-08-10T20:16:31+00:00\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\\\/\\\/schema.org\",\"@graph\":[{\"@type\":\"WebPage\",\"@id\":\"https:\\\/\\\/dornsife.usc.edu\\\/pzqin-lab\\\/dna-target-discrimination-by-crispr\\\/\",\"url\":\"https:\\\/\\\/dornsife.usc.edu\\\/pzqin-lab\\\/dna-target-discrimination-by-crispr\\\/\",\"name\":\"DNA Target Discrimination by CRISPR - 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