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روش CRATER : واکنش ترانفورماسیون با کارآیی بالا به واسطه کریسپر cas9

وش CRATER : واکنش ترانفورماسیون با کارآیی بالا به واسطه کریسپر/cas9

روشی نوین جهت ترانسفورماسیون انتخابی

سیستم کریسپر cas9 به واسطه فراهم سازی هدف گیری بی نظیر در توالی DNA مورد نظر، انقلابی در ویرایش ژنوم ایجاد نموده است. از این سیستم می توان جهت تسهیل انتخاب کارآی پلاسمید به مانند انتقال ژن مورد نظر به داخل وکتور پلاسمیدی استفاده نمود؛ چرا که باعث جداسازی محصولات ناخواسته پلاسمید پس از هضم توسط آنزیم محدود کننده و لایگیشن می شود.
در تحقیق پیش رو با استفاده از پروتئین های فلوئورسانس و رنگزا جهت نشان گذاری، نشان داده شده است که برش به واسطه کریسپر cas9 محصولات ناخواسته لایگیشن را حذف و کارآیی انتقال ژنی را برای توالی های مورد نظر از 20% تا 97% افزایش می دهد. روش CRATER: “واکنش ترانفورماسیون با کارآیی بالا به واسطه کریسپرcas9 ” راه کاری جدید، ارزان و ساده جهت تولید محصولات موردنظر می باشد.

CRISPR/Cas9-Assisted Transformation-Efficient Reaction

Molecular cloning is a fundamental technique in molecular biology to produce plasmid constructs. Several methods currently exist to minimize or select against unwanted plasmid products created during ligation of inserts into vectors, including cross-incompatible sticky ends, X-gal blue/white screening, dephosphorylation of backbone sticky ends, the addition of antibiotics, and agarose electrophoresis/gel extraction.

However, in special circumstances existing methods may be insufficient to quickly, cheaply, and effectively screen for specific cloning products. The use of antibiotics requires a host strain lacking resistance and the inclusion of genes conferring antibiotic resistance. Genes of interest may include restriction sites that would otherwise be used to create incompatible sticky ends. A plasmid vector also may simply not include multiple restriction sites with incompatible sticky ends. Unwanted byproducts are also difficult to control in situations where blunt ends are used.

We developed a new method for degrading unwanted ligation products using the Cas9 nuclease from Streptococcus pyogenes in a one-pot reaction, enhancing transformation efficiency from 20% up to 97% ± 3%. The Cas9 protein is a component of the clustered, regularly interspaced, short palindromic repeats (CRISPR) system. The CRISPR/CRISPR-associated (Cas) system provides bacteria with acquired immunity by incorporating fragments of foreign DNA and using the transcribed CRISPR-RNA (crRNA) to guide the cleavage of matching dsDNA sequences (Bhaya, Wiedenheft). In type II CRISPR systems, a ternary complex of Cas9, crRNA, and trans-activating crRNA (tracrRNA) binds to and cleaves dsDNA sequences that match the crRNA and include a short protospacer-adjacent motif (PAM) recognized by Cas9 (Gasiunas, Jinek). In type II systems, the crRNA and tracrRNA can be combined into a single guide-RNA (sgRNA) that is sufficient to lead Cas9 to its target (Jinek). Further, the PAM sequence recognized by the S. pyogenes Cas9 is only three nucleotides in length (NGG), allowing this system to be easily adapted to recognize and cut a desired sequence (Mojica).

With the knowledge that Cas9 can be used to cleave short (~24 bp) sequences, we investigated whether this system could be adapted to cleave unwanted ligation byproducts. We used the the RFP BioBrick plasmid (pSB1C3) as a starting vector and replaced the RFP insert with various genes of interest using restriction enzyme digestion and ligation, before transforming into Escherichia coli.

We then quantified insertion efficiency based on the presence of fluorescent proteins in colonies and culture. We show, for the first time to our knowledge, that Cas9 and sgRNAs can be used to increase molecular cloning efficiency by cleavage of specific ligation byproducts; we call this novel technique CRISPR/Cas9-assisted transformation-efficient reaction (CRATER).

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