{"id":8253,"date":"2020-11-09T13:27:57","date_gmt":"2020-11-09T10:27:57","guid":{"rendered":"http:\/\/blog.ulubat.org\/?p=8253"},"modified":"2020-11-09T13:30:19","modified_gmt":"2020-11-09T10:30:19","slug":"cascade-cas3-sistemi","status":"publish","type":"post","link":"https:\/\/blog.ulubat.org\/index.php\/genel\/cascade-cas3-sistemi\/","title":{"rendered":"CASCADE-CAS3 S\u0130STEM\u0130"},"content":{"rendered":"\n

2020 Nobel Kimya \u00d6d\u00fcl\u00fc: CRISPR<\/h3>\n\n\n\n

Yaz\u0131ma ba\u015flamadan ve konuya daha detayl\u0131 bakmadan \u00f6nce, inceledi\u011fim makalenin belki de \u2018ilham kayna\u011f\u0131\u2019 say\u0131labilecek y\u00f6nteminden genel olarak bahsetmek istiyorum: CRISPR. \u00c7o\u011fumuz belki de bu geli\u015fmenin ad\u0131n\u0131 ge\u00e7ti\u011fimiz haftalarda Kimya alan\u0131nda verilen Nobel \u00f6d\u00fcl\u00fc ile duydu, biraz\u0131m\u0131z da zaten biliyor ve takip ediyordu. Bu seneki Nobel Kimya \u00d6d\u00fcl\u00fc, CRISPR tekni\u011finin \u00fczerinde \u00e7al\u0131\u015fan ve onu geli\u015ftiren bilim insanlar\u0131na verildi. Bu durum ve halihaz\u0131rda y\u00fcr\u00fct\u00fclmekte olan bir\u00e7ok ara\u015ft\u0131rma, bizlere gen editleme tekniklerinin potansiyelini ve \u00f6nemini g\u00f6sterdi. Asl\u0131nda bakterilerden esinlenerek laboratuvarlarda yayg\u0131n bir \u015fekilde kullan\u0131lan bir teknik haline gelen CRISPR; ayn\u0131 zamanda iyile\u015ftirilmeye, h\u0131zland\u0131r\u0131lmaya ve belki de daha da spesifik hale getirilmeye a\u00e7\u0131k olabilecek olan bir y\u00f6ntemdir. \u00c7al\u0131\u015fmalarda yo\u011fun olarak kullan\u0131lan CRISPR-Cas9 sistemi, spesifik bir bakteri enzimi kullan\u0131larak \u00e7al\u0131\u015fmaktad\u0131r. Ancak Cas9, benzer i\u015flevleri yerine getirmek i\u00e7in kullan\u0131labilecek tek enzim de de\u011fildir. University of California, San Francisco\u2019daki ara\u015ft\u0131rmac\u0131lar da Cas9\u2019dan daha iyi alternatifler kullan\u0131labilir mi sorusuna cevap aray\u0131p ara\u015ft\u0131rmalar\u0131n\u0131 y\u00fcr\u00fctm\u00fc\u015f ve bunu test etmek i\u00e7in de Cas3 enzimini kullanmaya karar vermi\u015flerdir. 19 Ekim 2020 tarihinde Nature Methods\u2019da yay\u0131mlanan makalenin ara\u015ft\u0131rmac\u0131lar\u0131ndan Joseph Bondy-Denomy, kulland\u0131klar\u0131 enzimin hedefini bulmas\u0131n\u0131n \u00fczerine DNA \u00fczerinde ilerleyip bu k\u0131sm\u0131 Pac-Man karakterine benzer bir bi\u00e7imde kesti\u011fini belirtmi\u015ftir\u00a0(CRISPR Meets Pac-Man: New DNA Cut-and-Paste Tool Enables Bigger Gene Edits — ScienceDaily<\/em>, 2020)<\/p>\n\n\n\n

\"Image
\u015eekil 1.\u00a0
(https:\/\/medium.com\/@lana.bozanic\/controlling-evolution-with-gene-editing-5e8a1edf322a<\/a>)\u00a0<\/figcaption><\/figure>\n\n\n\n

Farkl\u0131 CRISPR Sistemleri<\/h3>\n\n\n\n

CRISPR sistemleri, halihaz\u0131rda \u00f6karyot ve prokaryot canl\u0131larda gen d\u00fczenleme s\u00fcre\u00e7lerini m\u00fcmk\u00fcn k\u0131lmaktad\u0131r ancak baz\u0131 sistemlerde kullan\u0131lan Cas-9 ve Cas-12 enzimlerinin b\u00fcy\u00fck delesyonlar yapma kabiliyetleri s\u0131n\u0131rl\u0131d\u0131r\u00a0(Cs\u00f6rg\u0151 et al., 2020). Sahip olduklar\u0131 alt yap\u0131 say\u0131s\u0131na g\u00f6re CRISPR-Cas sistemleri iki s\u0131n\u0131fa ayr\u0131lmaktad\u0131r: S\u0131n\u0131f 1 (birden fazla par\u00e7a) ve S\u0131n\u0131f 2 (bir par\u00e7a) ve s\u0131n\u0131f 1 CRISPR sistemleri do\u011fada daha da s\u0131k g\u00f6r\u00fclmektedir\u00a0(Koonin & Makarova, 2019). Genellikle literat\u00fcrdeki CRISPR \u00fczerine yap\u0131lm\u0131\u015f olan \u00e7al\u0131\u015fmalar, tek par\u00e7al\u0131 s\u0131n\u0131f 2 CRISPR sistemlerine odaklanm\u0131\u015ft\u0131r\u00a0(Barrangou & Doudna, 2016)\u00a0ancak birka\u00e7 par\u00e7al\u0131 olan s\u0131n\u0131f 1 sistemleri de \u00e7al\u0131\u015fmalarda \u00e7ok \u00f6nemli geli\u015fmeler ya\u015fanmas\u0131n\u0131 sa\u011flayabilir. Cas-3, ayn\u0131 zamanda sahip oldu\u011fu par\u00e7alar sayesinde b\u00fcy\u00fck gen par\u00e7alar\u0131n\u0131n kesilmesinde de s\u0131n\u0131f 2 enzimlere k\u0131yasla daha verimli bir \u015fekilde kullan\u0131labilir\u00a0(Cs\u00f6rg\u0151 et al., 2020). Bu d\u00fc\u015f\u00fcnceden yola \u00e7\u0131karak da\u00a0Cs\u00f6rg\u0151 ve ekibi, P. aeruginosa\u2019dan al\u0131n\u0131p hedefe g\u00f6re yeniden \u015fekillendirilen bir CRISPR sistemini test etmi\u015flerdir. Bahsi ge\u00e7en Cascade-Cas3 sistemiyle yap\u0131lan genom baz\u0131ndaki de\u011fi\u015fikliklerin, g\u00fcn\u00fcm\u00fczde ba\u015fka teknikler kullan\u0131larak yap\u0131lmas\u0131 m\u00fcmk\u00fcn g\u00f6z\u00fckmemektedir\u00a0(Cs\u00f6rg\u0151 et al., 2020). Bu sistemde Cas proteinleri ve crRNA\u2019n\u0131n bir araya gelerek olu\u015fturdu\u011fu Cascade kompleksi\u00a0(Brouns et al., 2008), hem belli bir DNA dizisinin hedeflenmesini m\u00fcmk\u00fcn k\u0131lmakta hem de o noktaya ula\u015f\u0131ld\u0131\u011f\u0131nda (a\u015fa\u011f\u0131daki resimde g\u00f6sterildi\u011fi gibi) Cas3 enzimini \u00e7a\u011f\u0131rmaktad\u0131r\u00a0(Rath et al., 2015).\u00a0<\/p>\n\n\n\n

\"The
\u015eekil 2, \u00e7e\u015fitli CRISPR-Cas sistemlerinin diyagramlar \u015feklinde temsili g\u00f6sterimi.\u00a0
(Pickar-Oliver & Gersbach, 2019)<\/figcaption><\/figure>\n\n\n\n

Cas3 Niye \u00d6zel?<\/h3>\n\n\n\n

CRISPR-Cas3 sistemleri, insan genomunda uzun hedef k\u0131s\u0131mlar\u0131 kesebilme kabiliyetleriyle Cas9 gibi sistemlerden ayr\u0131lmaktad\u0131r (CRISPR-Cas3 Innovation Holds Promise for Disease Cures, Advancing Science — ScienceDaily<\/em>, 2019). Bu \u00f6zelli\u011finin, onun gelecekte kullan\u0131m\u0131n\u0131 art\u0131raca\u011f\u0131 d\u00fc\u015f\u00fcn\u00fclmektedir. \u00d6rne\u011fin, halk sa\u011fl\u0131\u011f\u0131nda \u00f6nemli say\u0131lan ektopik vir\u00fcslerin hedeflenmesinde kullan\u0131labilir. CRISPR-Cas3 \u00fczerine \u00e7al\u0131\u015fan bir di\u011fer bilim insan\u0131, Ailong Ke, \u00fczerine \u00e7al\u0131\u015ft\u0131klar\u0131 sistemlerin b\u00f6yle vir\u00fcsleri \u00e7ok spesifik bir \u015fekilde hedefleyip silebilece\u011fini, dolay\u0131s\u0131yla da ilerde viral hastal\u0131klar\u0131n tedavisinde kullan\u0131labilece\u011fini dile getirmi\u015ftir (CRISPR-Cas3 Innovation Holds Promise for Disease Cures, Advancing Science — ScienceDaily<\/em>, 2019). <\/p>\n\n\n\n

Bunun haricinde, Cas3 sistemleri DNA\u2019n\u0131n kodlamayan k\u0131s\u0131mlar\u0131n\u0131 da ara\u015ft\u0131rmak i\u00e7in elveri\u015fli olabilir. Genetik alan\u0131nda \u00e7ok ilerlemi\u015f olsak da hala bu k\u0131s\u0131mlar\u0131n i\u015flevlerine dair \u00e7ok derin bir bilgi birikimimiz bulunmamaktad\u0131r. CRISPR-Cas3 de kodlamayan k\u0131s\u0131mlar i\u00e7in genomu taray\u0131p bunlarda de\u011fi\u015fiklikler yaparak veya onlar\u0131 silerek i\u015flevlerinin model organizmalarda ara\u015ft\u0131r\u0131lmas\u0131n\u0131 m\u00fcmk\u00fcn k\u0131labilir. <\/p>\n\n\n\n

Yine de \u00e7o\u011fu deneyde oldu\u011fu gibi, Cascade-Cas3 sistemini inceleyen ara\u015ft\u0131rmac\u0131lar da y\u00f6ntemlerinin planland\u0131\u011f\u0131 kadar verimli \u00e7al\u0131\u015fmas\u0131na engel olabilecek etkenleri de g\u00f6zetim alt\u0131na almay\u0131 s\u00fcrd\u00fcrm\u00fc\u015flerdir. Bunlardan biri, CRISPR-Cas3 sisteminin yapt\u0131\u011f\u0131 kesimin s\u0131n\u0131rlar\u0131n\u0131n bazen kesin olarak belirlenememesidir\u00a0(CRISPR-Cas3 Innovation Holds Promise for Disease Cures, Advancing Science — ScienceDaily<\/em>, 2019). A\u015f\u0131lmas\u0131 gereken engellerden bir di\u011feri de baz\u0131 profajlarda \u00fcretilebilen anti-CRISPR proteinleridir. Ara\u015ft\u0131rmac\u0131lar, bakteriyofajda AcrIC1 adl\u0131 bir anti-CRISPR proteini bulunuyorsa bakterinin CRISPR-Cas3 sisteminin aktivitesini engelleyebildi\u011fini, ancak Cas9 inhibit\u00f6r\u00fcn\u00fcn ayn\u0131 aktiviteyi ger\u00e7ekle\u015ftirmedi\u011fini kaydetmi\u015flerdir. Anti-CRISPR proteinlerine kar\u015f\u0131 bir \u00f6nlem olarak da \u2018anti-anti-CRISPR\u2019 stratejisi denenmi\u015f, CRISPR sistemlerini engelleyen proteinlerin sentezini bask\u0131layan ba\u015fka bir protein kullan\u0131lmaya \u00e7al\u0131\u015f\u0131lm\u0131\u015ft\u0131r. Dolay\u0131s\u0131yla da anti-CRISPR proteinlerini bask\u0131layan etmenlerin CRISPR-Cas sistemlerinin etkinli\u011fini daha verimli hale getirecek oldu\u011fu d\u00fc\u015f\u00fcn\u00fclm\u00fc\u015ft\u00fcr. Ba\u015fka bir bak\u0131\u015f a\u00e7\u0131s\u0131ndan bakarak ara\u015ft\u0131rmay\u0131 y\u00fcr\u00fcten ara\u015ft\u0131rmac\u0131lardan Joseph Bondy-Denomy; CRISPR\u2019a kar\u015f\u0131 fajlar taraf\u0131ndan geli\u015ftirilmi\u015f bu tip mekanizmalar\u0131n, insanlarda kullan\u0131lan Cas tedavilerinde tespit edilen yan etkileri, zarara yol a\u00e7madan \u00f6nce durdurmak i\u00e7in de kullan\u0131labilece\u011fini dile getirmi\u015ftir\u00a0(CRISPR Meets Pac-Man: New DNA Cut-and-Paste Tool Enables Bigger Gene Edits — ScienceDaily<\/em>, 2020).\u00a0\u00a0<\/p>\n\n\n\n

\"\"
\u015eekil 3, mavi ile temsil edilmi\u015f Cascade proteini ve ona ba\u011fl\u0131 CRISPR RNA\u2019s\u0131 (k\u0131rm\u0131z\u0131).\u00a0
(Goodsell, 2015)<\/figcaption><\/figure>\n\n\n\n

\u00c7al\u0131\u015fmalar bizi nereye g\u00f6t\u00fcr\u00fcyor?<\/h3>\n\n\n\n

Bu \u00e7al\u0131\u015fma, CRISPR-Cas3 sistemlerinin \u00f6karyot h\u00fccrelerinde, \u00f6zellikle tekrar eden ve uzun k\u0131s\u0131mlar\u0131n hedef al\u0131nmas\u0131ndaki potansiyelini \u00f6ne koymu\u015f, ayn\u0131 zamanda bu sistemin geri kalabilece\u011fi alanlardan da bahsetmi\u015ftir. Anlatt\u0131klar\u0131m ve genel olarak CRISPR hakk\u0131nda daha detayl\u0131 bilgi almak isterseniz kaynak\u00e7amda belirtti\u011fim makalelere ula\u015fabilirsiniz. Hala geli\u015fmekte olan ve i\u00e7inde \u00e7ok yeni teknikler bar\u0131nd\u0131ran bu konuda daha fazla ara\u015ft\u0131rmaya ihtiyac\u0131m\u0131z var. DNA\u2019da daha b\u00fcy\u00fck k\u0131s\u0131mlar\u0131n kesilebilmesi, daha \u00e7ok genin i\u015flevini anlayabilmemizi de beraberinde getirecektir. Ayn\u0131 zamanda, bu tarz i\u015flevsel bir sistem bakterilerde metabolizma de\u011fi\u015fikliklerine neden olabilen gen par\u00e7alar\u0131n\u0131n da ay\u0131klanabilmesini sa\u011flayarak (CRISPR Meets Pac-Man: New DNA Cut-and-Paste Tool Enables Bigger Gene Edits — ScienceDaily<\/em>, n.d.) biyoteknoloji \u00e7al\u0131\u015fmalar\u0131n\u0131 da ileri ta\u015f\u0131yabilir. <\/p>\n\n\n\n

Kaynak\u00e7a <\/h3>\n\n\n\n

Barrangou, R., & Doudna, J. A. (2016). Applications of CRISPR technologies in research and beyond. Nature Biotechnology<\/em>. https:\/\/doi.org\/10.1038\/nbt.3659<\/p>\n\n\n\n

Brouns, S. J. J., Jore, M. M., Lundgren, M., Westra, E. R., Slijkhuis, R. J. H., Snijders, A. P. L., Dickman, M. J., Makarova, K. S., Koonin, E. V., & Van Der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science<\/em>,321<\/em>(5891), 960\u2013964. https:\/\/doi.org\/10.1126\/science.1159689<\/p>\n\n\n\n

CRISPR-Cas3 innovation holds promise for disease cures, advancing science — ScienceDaily<\/em>. (2019). https:\/\/www.sciencedaily.com\/releases\/2019\/04\/190411172519.htm<\/p>\n\n\n\n

CRISPR meets Pac-Man: New DNA cut-and-paste tool enables bigger gene edits — ScienceDaily<\/em>. (n.d.). Retrieved November 6, 2020, from https:\/\/www.sciencedaily.com\/releases\/2020\/10\/201020105551.htm<\/p>\n\n\n\n

Cs\u00f6rg\u0151, B., Le\u00f3n, L. M., Chau-Ly, I. J., Vasquez-Rifo, A., Berry, J. D., Mahendra, C., Crawford, E. D., Lewis, J. D., & Bondy-Denomy, J. (2020). A compact Cascade\u2013Cas3 system for targeted genome engineering. Nature Methods<\/em>. https:\/\/doi.org\/10.1038\/s41592-020-00980-w<\/p>\n\n\n\n

Goodsell, D. S. (2015). Cascade and CRISPR. RCSB Protein Data Bank<\/em>. https:\/\/doi.org\/10.2210\/rcsb_pdb\/mom_2015_1<\/p>\n\n\n\n

Koonin, E. V., & Makarova, K. S. (2019). Origins and evolution of CRISPR-Cas systems. In Philosophical Transactions of the Royal Society B: Biological Sciences<\/em> (Vol. 374, Issue 1772). Royal Society Publishing. https:\/\/doi.org\/10.1098\/rstb.2018.0087<\/p>\n\n\n\n

Pickar-Oliver, A., & Gersbach, C. A. (2019). The next generation of CRISPR\u2013Cas technologies and applications. In Nature Reviews Molecular Cell Biology<\/em>. https:\/\/doi.org\/10.1038\/s41580-019-0131-5<\/p>\n\n\n\n

Rath, D., Amlinger, L., Hoekzema, M., Devulapally, P. R., & Lundgren, M. (2015). Efficient programmable gene silencing by Cascade. Nucleic Acids Research<\/em>, 43<\/em>(1), 237\u2013246. https:\/\/doi.org\/10.1093\/nar\/gku1257<\/p>\n","protected":false},"excerpt":{"rendered":"

2020 Nobel Kimya \u00d6d\u00fcl\u00fc: CRISPR Yaz\u0131ma ba\u015flamadan ve konuya daha detayl\u0131 bakmadan \u00f6nce, inceledi\u011fim makalenin belki de \u2018ilham kayna\u011f\u0131\u2019 say\u0131labilecek<\/p>\n","protected":false},"author":273,"featured_media":8255,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[1],"tags":[95,1322,270,149,115,76,453],"acf":[],"views":865,"_links":{"self":[{"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/posts\/8253"}],"collection":[{"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/users\/273"}],"replies":[{"embeddable":true,"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/comments?post=8253"}],"version-history":[{"count":5,"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/posts\/8253\/revisions"}],"predecessor-version":[{"id":8260,"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/posts\/8253\/revisions\/8260"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/media\/8255"}],"wp:attachment":[{"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/media?parent=8253"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/categories?post=8253"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/blog.ulubat.org\/index.php\/wp-json\/wp\/v2\/tags?post=8253"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}