Turkiye Klinikleri Journal of Medical Sciences

.: REVIEW
CRISPR-Cas Fonksiyonel Genetik Tarama: Geleneksel Derleme
CRISPR-Cas Functional Genetic Screening: Traditional Review
Hale GÜLER KARAa,b, Buket KOSOVAb, Eda DOĞANb, Vildan BOZOK ÇETİNTAŞb, Şerif ŞENTÜRKc
aHarran Üniversitesi Tıp Fakültesi, Tıbbi Biyoloji ABD, Şanlıurfa, Türkiye
bEge Üniversitesi Tıp Fakültesi, Tıbbi Biyoloji ABD, İzmir, Türkiye
cDokuz Eylül Üniversitesi İzmir Uluslararası Biyotıp ve Genom Enstitüsü, İzmir, Türkiye
Turkiye Klinikleri J Med Sci. 2022;42(4):311-22
doi: 10.5336/medsci.2022-88507
Article Language: TR
Full Text
ÖZET
 Prokaryotlara ait savunma mekanizması bileşenleri kullanılarak geliştirilen düzenli aralıklarla bölünmüş kısa palindromik tekrar kümeleri [clustered regularly interspaced palindromic repeats (CRISPR)-Cas] sistemi hedefe özgü gen düzenleme aracı olarak in vitro ve in vivo çalışmalarda yaygın olarak kullanılmaktadır. CRISPR-Cas sisteminin kullanım alanları arasında gen silme, gen ekleme, baz düzeltme, gen ifadesi düzenleme, genomik lokusları görüntüleme, epigenetik düzenleme ve diagnostik analizler yer almaktadır. Bu sistemin güçlü özellikleri arasında bulunan esnekliği, çok yönlülüğü ve kolay uygulanabilirliği kullanım alanlarının giderek artmasına yol açmıştır. Son yıllarda, genlerin biyolojik süreçler ve hücresel işlevlerdeki rollerini aydınlatmak için de CRISPR-Cas9 temelli fonksiyonel genetik tarama çalışmaları yapılmaktadır. CRISPR taramaları, başta kanser olmak üzere, hastalığa bağlı olarak hücrelerin hayatta kalabilmeleri için gerekli olan, hastalık gelişiminden sorumlu genetik faktörlerin tanımlanması ve fonksiyonlarının belirlenmesi için kullanılmaktadır. Amaca uygun seçilmiş gRNA kütüphanesinin hazırlanması ve çoğaltılması, ilgili plazmide klonlama, lentivirüslere paketleme ve hücrelere lentiviral transdüksiyon, fenotipik seçilim, yeni nesil DNA dizi analizi ve biyoinformatik analiz aşamalarından oluşan CRISPR-Cas temelli taramalar, fonksiyonel genetik çalışmalarının yüksek verimlilik ve doğrulukta yapılabilmesini mümkün kılmıştır. Patojen-konakçı etkileşimlerinin araştırılması, hastalıkların moleküler mekanizmalarının ortaya konması, yeni tedavi hedeflerinin bulunması ve tedavi direncinde rol oynayan genetik faktörlerin belirlenmesi gibi farklı amaçlara yönelik tarama çalışmaları da yapılabilmektedir. Bu derlemede, farklı CRISPR-Cas sistemleri kısaca özetlendikten sonra genetik tarama çalışmalarının temel mantığı, uygulanışı ve kullanım alanları güncel literatürden seçilen örneklerle tartışılacaktır.

Anahtar Kelimeler: CRISPR-Cas; CRISPR tarama; fonksiyonel genetik tarama; gRNA kütüphanesi
ABSTRACT
 The clustered regularly interspaced palindromic repeats (CRISPR)-associated Cas system, derived from the defence mechanism components of prokaryotes, is frequently used as a target specific gene editing tool in in vitro and in vivo studies. Uses of the CRISPR-Cas system include gene deletion, gene insertion, base editing, gene expression editing, genomic locus imaging, epigenetic editing, and diagnostic analysis. Due to the strengths of the system such as flexibility, versatility and easy application, its usage areas have gradually increased. In recent years, CRISPR-Cas9 based functional genetic screen studies have been carried out to understand the roles of genes in the biological processes and cellular functions. CRISPR screens are used to identify genetic factors responsible for disease development and determine their functions, which are necessary for the survival of cells due to disease, especially cancer. CRISPR-Cas9 screens consisting of desing and amplification of the selected gRNA library, cloning in the relevant plasmid, packaging into lentiviruses and lentiviral transduction into cells, phenotypic selection, next generation DNA sequence analysis and bioinformatics analysis, has allowed functional genetic studies that can be performed with high-throughput and high-efficiency. Screening studies can be carried out for different purposes such as investigating pathogen-host interactions, revealing the molecular mechanisms of diseases, identifying novel therapeutic targets and revealing factors that play a key role in treatment resistance. In this review, after a short summary of different CRISPRCas systems the basic principles of genetic screening studies, their application and areas of usage will be discussed with selected examples from the current literature.

Keywords: CRISPR-Cas; CRISPR screen; functional genetic screen; gRNA library
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- Providing scientific articles, books and informative publications for health industry.

- Providing structural, statistical and editorial support to article preparation stage for scientific journals.

4. GENERAL PROVISIONS

4.1. "Turkiye Klinikleri" is completely free to determine which of the services and contents provided in the "SITE" will be charged.

4.2. People benefiting from the services provided by "Turkiye Klinikleri" and using the website can use the "SITE" only according to the law and only for personal reasons. Users have the criminal and civil liability for every process and action they take in the "SITE". Every USER agrees, declares and undertakes that they will not proceed by any function or action infringement of rights of "Turkiye Klinikleri"s and/or other third parties', they are the exclusive right holder on usage, processing, storage, made public and revealing any written, visual or auditory information reported to Turkiye Klinikleri" and/or "SITE" to the third parties. "USER" agrees and undertakes that s/he will not duplicate, copy, distribute, process, the pictures, text, visual and auditory images, video clips, files, databases, catalogs and lists within the "SITE", s/he will not be using these actions or with other ways to compete with "Turkiye Klinikleri", directly or indirectly.

4.3. The services provided and the context published within the "SITE" by third parties is not under the responsibility of "Turkiye Klinikleri", institutions collaborated with "Turkiye Klinikleri", "Turkiye Klinikleri" employee and directors, "Turkiye Klinikleri" authorized salespeople. Commitment to accuracy and legality of the published information, context, visual and auditory images provided by any third party are under the full responsibility of the third party. "Turkiye Klinikleri" does not promise and guarantee the safety, accuracy and legality of the services and context provided by a third party.

4.4. "USER"s cannot act against "Turkiye Klinikleri", other "USER"s and third parties by using the "SITE". "Turkiye Klinikleri" has no direct and/or indirect responsibility for any damage a third party suffered or will suffer regarding "USER"s actions on the "SITE" against the rules of the hereby "Terms of Use" and the law.

4.5. "USER"s accept and undertake that the information and context they provided to the "SITE" are accurate and legal. "Turkiye Klinikleri" is not liable and responsible for promising and guaranteeing the verification of the information and context transmitted to "Turkiye Klinikleri" by the "USER"s, or uploaded, changed and provided through the "SITE" by them and whether these information are safe, accurate and legal.

4.6. "USER"s agree and undertake that they will not perform any action leading to unfair competition, weakening the personal and commercial credit of "Turkiye Klinikleri" and a third party,  encroaching and attacking on personal rights within the "SITE" in accordance with the Turkish Commercial Code Law.

4.7. "Turkiye Klinikleri" reserves the right to change the services and the context within the "SITE"  anytime. "Turkiye Klinikleri" may use this right without any notification and timelessly. "USER"s have to make the changes and/or corrections "Turkiye Klinikleri" required immediately. Any changes and/or corrections that are required by "Turkiye Klinikleri", may be made by "Turkiye Klinikleri" when needed. Any harm, criminal and civil liability resulted or will result from changes and/or corrections required by "Turkiye Klinikleri" and were not made on time by the "USER"s belongs completely to the users.

4.8. "Turkiye Klinikleri" may give links through the "SITE" to other websites and/or "CONTEXT"s and/or folders that are outside of their control and owned and run by third parties. These links are provided for ease of reference only and do not hold qualification for support the respective web SITE or the admin or declaration or guarantee for the information inside. "Turkiye Klinikleri" does not hold any responsibility over the web-sites connected through the links on the "SITE", folders and context, the services or products on the websites provided through these links or their context.

4.9. "Turkiye Klinikleri" may use the information provided to them by the "USERS" through the "SITE" in line with the terms of the "PRIVACY POLICY" and "USER CONTRACT". It may process the information or classify and save them on a database. "Turkiye Klinikleri" may also use the USER's or visitor's identity, address, e-mail address, phone number, IP number, which sections of the "SITE" they visited, domain type, browser type, date and time information to provide statistical evaluation and customized services.

5. PROPRIETARY RIGHTS

5.1. The information accessed through this "SITE" or provided by the users legally and all the elements (including but not limited to design, text, image, html code and other codes) of the "SITE" (all of them will be called as studies tied to "Turkiye Klinikleri"s copyrights) belongs to "Turkiye Klinikleri". Users do not have the right to resell, process, share, distribute, display or give someone permission to access or to use the "Turkiye Klinikleri" services, "Turkiye Klinikleri" information and the products under copyright protection by "Turkiye Klinikleri". Within hereby "Terms of Use" unless explicitly permitted by "Turkiye Klinikleri" nobody can reproduce, process, distribute or produce or prepare any study from those under "Turkiye Klinikleri" copyright protection.

5.2. Within hereby "Terms of Use", "Turkiye Klinikleri" reserves the rights for "Turkiye Klinikleri" services, "Turkiye Klinikleri" information, the products associated with "Turkiye Klinikleri" copyrights, "Turkiye Klinikleri" trademarks, "Turkiye Klinikleri" trade looks or its all rights for other entity and information it has through this website unless it is explicitly authorized by "Turkiye Klinikleri".

6. CHANGES IN THE TERMS OF USE

"Turkiye Klinikleri" in its sole discretion may change the hereby "Terms of Use" anytime announcing within the "SITE". The changed terms of the hereby "Terms of Use" will become valid when they are announced. Hereby "Terms of Use" cannot be changed by unilateral declarations of users.

7. FORCE MAJEURE

"Turkiye Klinikleri" is not responsible for executing late or never of this hereby "Terms of Use", privacy policy and "USER Contract" in any situation legally taken into account as force majeure. Being late or failure of performance or non-defaulting of this and similar cases like this will not be the case from the viewpoint of "Turkiye Klinikleri", and "Turkiye Klinikleri" will not have any damage liability for these situations. "Force majeure" term will be regarded as outside of the concerned party's reasonable control and any situation that "Turkiye Klinikleri" cannot prevent even though it shows due diligence. Also, force majeure situations include but not limited to natural disasters, rebellion, war, strike, communication problems, infrastructure and internet failure, power cut and bad weather conditions.

8. LAW AND AUTHORISATION TO FOLLOW

Turkish Law will be applied in practicing, interpreting the hereby "Terms of Use" and managing the emerging legal relationships within this "Terms of Use" in case of finding element of foreignness, except for the rules of Turkish conflict of laws. Ankara Courts and Enforcement Offices are entitled in any controversy happened or may happen due to hereby contract.

9. CLOSING AND AGREEMENT

Hereby "Terms of Use" come into force when announced in the "SITE" by "Turkiye Klinikleri". The users are regarded to agree to hereby contract terms by using the "SITE". "Turkiye Klinikleri" may change the contract terms and the changes will be come into force by specifying the version number and the date of change on time it is published in the "SITE".

 

30.03.2014

Privacy Policy

We recommend you to read the terms of use below before you visit our website. In case you agree these terms, following our rules will be to your favor. Please read our Terms of Use thoroughly.

www.turkiyeklinikleri.com website belongs to Ortadoğu Advertisement Presentation Publishing Tourism Education Architecture Industry and Trade Inc. and is designed in order to inform physicians in the field of health

www.turkiyeklinikleri.com cannot reach to user’s identity, address, service providers or other information. The users may send this information to the website through forms if they would like to. However, www.turkiyeklinikleri.com may collect your hardware and software information. The information consists of your IP address, browser type, operating system, domain name, access time, and related websites. www.turkiyeklinikleri.com cannot sell the provided user information (your name, e-mail address, home and work address, phone number) to the third parties, publish it publicly, or keep it in the website. Gathered information has a directing feature to be a source for the website’s visitor profile, reporting and promotion of the services.

www.turkiyeklinikleri.com uses the taken information:

-To enhance, improve and maintain the quality of the website

-To generate visitor’s profile and statistical data

-To determine the tendency of the visitors on using our website

-To send print publications/correspondences

-To send press releases or notifications through e-mail

-To generate a list for an event or competition

By using www.turkiyeklinikleri.com you are considered to agree that;

-Ortadoğu Advertisement Presentation Publishing Tourism Education Architecture Industry and Trade Inc. cannot be hold responsible for any user’s illegal and immoral behavior,

-Terms of use may change from time to time,

-It is not responsible for other websites’ contents it cannot control or the harms they may cause although it uses the connection they provided.

Ortadoğu Advertisement Presentation Publishing Tourism Education Architecture Industry and Trade Inc. may block the website to users in the following events:

-Information with wrong, incomplete, deceiving or immoral expressions is recorded to the website,

-Proclamation, advertisement, announcement, libelous expressions are used against natural person or legal identity,

-During various attacks to the website,

-Disruption of the website because of a virus.

Written, visual and audible materials of the website, including the code and the software are under protection by legal legislation.

Without the written consent of Ortadoğu Advertisement Presentation Publishing Tourism Education Architecture Industry and Trade Inc. the information on the website cannot be downloaded, changed, reproduced, copied, republished, posted or distributed.

All rights of the software and the design of the website belong to Ortadoğu Advertisement Presentation Publishing Tourism Education Architecture Industry and Trade Inc.

Ortadoğu Advertisement Presentation Publishing Tourism Education Architecture Industry and Trade Inc. will be pleased to hear your comments about our terms of use. Please share the subjects you think may enrich our website or if there is any problem regarding our website.

info@turkiyeklinikleri.com