كريسپر
كريسپر CRISPR (clustered regularly interspaced short palindromic repeats) هوموضع صبغوي يحتوي على عدة تكرارات مباشرة قصيرة تم العثور عليها في جينوم ما يقرب من 40٪ من متسلسلة البكتيريا و90٪ من متسلسلة العتيقة.
يوجد الكريسپر في 40% من جينومات الجراثيم المتسلسلة 90% من جينومات العتائق المتسلسلة.
عادة ما يرتبط الكريسپر مع جينات كاس التي ترمز للپروتينات المرتبطة بالكريسپر. نظام كريسپر/كاس هوالجهاز المناعي لبدائيات النوى الذي يمنح مقاومة المواد الجينية الخارجية مثل الپلازميدات والعاثيات ويفور شكلاً من أشكال المناعة المكتسبة. فواصل الكريسپر تتعهد وتبتر هذه المواد الجينية الغريبة بطريقة مماثلة لتداخل الحمض النووي الربيبوزي في العضيات حقيقية النوى.
منذ 2013، يستخدم نظام كريسپر/كاس في في تحرير الجينوم (الإضافة، التعطيل، تغيير تسلسل بعض الجينات) وتنظيم الجينات في الأنواع ضمن شجرة الحياة. بتقديم پروتين كاس9 والدليل المناسب للحمض النووي الربيوزي داخل الخلية، يمكن بتر جينوم العضية في المكان المطلوب.
يمكن استعمال كريسپر لبناء بواعث جينوم موجة الرنا قادرة على تغيير جينومات جميع المجموعات السكانية.
نسخة مبسطة من نظام كريسپر/كاس، كريسپر/كاس9، تم تعديلها إلى جينومات التحرير. من خلال وضع نوكلياز كاس9 المركب مع مرشد الرنا (gRNA) داخل الخلية، يمكن بتر جينوم الخلية عند الموضوع المطلوب، مما يسمح بإزالة الجينات القائمة و/أوإضافة جينات جديدة. يتوافق مركب كاس9-دليل الرنا مع مركب كاس ثلاثة crRNA كما هومشروح في المخطط أعلاه.
يمكن استعمال تقنيات تحرير جينوم كريسپر/كاس في الكثير من التطبيقات، ومنها الطب وتحسين بذور الحاصلات الزراعية. استخدام مركب كريسپر/كاس9-دليل الرنا في تحرير الجينوم كان خيار الاتحاد الأمريكي لتقدم العلوم لاختراق العام في 2015. ظهر مخاوف أخلاقية حيوية حول احتمال استخدام كريسپر في تحرير الخط الجنسي.
فيخمسة يناير 2018، أعرب الفهماء عن حتى كاس9 موجود ضمن أكثر من 65% من الأشخاص، مما يعني توافر الفرصة لدى هؤلاء الأشخاص للاستفادة من تطبيقات كريسپر في العلاج والوقاية من الأمراض، ولا يعني هذا حتى النسبة الباقية الذين توجد لديهم أجسام مناعية تعوق عمل كريسپر لا يمكنهم الاستفادة من هذه التطبيقات، لكن التأثيرات العلاجية قد تكون أقل، وفي بعض الحالات قد تؤدي إلى ظهور آثار سمية كبيرة لدى السقمى.
| كرسپر CRISPR | |
|---|---|
| Structure of crRNA-guided E. coli Cascade complex (Cas, blue) bound to single-stranded DNA (orange). | |
| المعهدات | |
| العضية | |
| الرمز | ? |
| بيانات أخرى | |
التاريخ
كرسپر هوجزء من عملية بكتيرية تحدث طبيعياً. فالبكتريا قد تضم دنا أجنبي وحتى قد تفترس دنا معطوب من بيئتها.
التكرارات المتعقدة تم وصفهم لأول مرة في 1987 للبكتريا إشريشيا كولاي على يد الباحث في جامعة اوساكا يوشي‌زومي إيشي‌نو، ولكن آنذاك فإن وظيفتهم لم تكن معروفة. وقد لوحظت التكرارات، بشكل مستقل من قِبل الباحث الإسباني فرانشسكوموخيكا في 1993. وسيسميهم لاحقاً "short regularly spaced repeats" (SRSR) بعد حتى عثر عليهم في الكثير من الميكروبات الأخرى. وفي عام 2000، تم التعهد على تكرارات مماثلة في بكتريا أخرى وفي عتائق أيضاً. وقد تغير اسم SRSR إلى كريسپر CRISPR في 2002 بناء على اقتراح من موخيكا. وقد اقتـُرِح حتى كريسپر هي المسئولة عن توليد المناعة المتكيفة في الميكروبات. الورقة البحثية التي وصفت تلك الفكرة رُفِضت في البداية من عدد من الدوريات البارزة. الأوراق البحثية التي اقترحت فرضيات مماثلة من جيل ڤرنووبشكل مستقل، ألكسندر بولوتان قابلت رفضاً مماثلاً قبل حتى تُنشر بعد لأي.
مخطات عشرات الآلاف من guide RNA متوفرة.
أسلاف تحرير الجين
بنية المواضع
التكرار والفواصل
جينات كاس وأنواع كريسپر الفرعية
| نوع كاس | جين التوقيع | الوظيفة | المصدر |
|---|---|---|---|
| I | Cas3 | Single-stranded DNA nuclease (HD domain) and ATP-dependent helicase | |
| IA | Cas8a | Subunit of the interference module | |
| IB | Cas8b | ||
| IC | Cas8c | ||
| ID | Cas10d | contains a domain homologous to the palm domain of nucleic acid polymerases and nucleotide cyclases | |
| IE | Cse1 | ||
| IF | Csy1 | لم يحدد | |
| II | Cas9 | Nucleases RuvC and HNH together produce DSBs, and separately can produce single-strand breaks. Ensures the acquisition of functional spacers during adaptation. | |
| IIA | Csn2 | لم يحدد | |
| IIB | Cas4 | لم يحدد | |
| IIC | Characterized by the absence of either Csn2 or Cas4 | ||
| III | Cas10 | Homolog of Cas10d and Cse1 | |
| IIIA | Csm2 | لم يحدد | |
| IIIB | Cmr5 | Not Determined |
الآلية
CRISPR-Cas immunity is a natural process of bacteria and archaea. CRISPR-Cas prevents bacteriophage infection, conjugation and natural transformation by degrading foreign nucleic acids that enter the cell.
الحصول على الفواصل داخل مواضع الكريسپر
فترة التداخل
| الپروتين المرتبط بكريسپر | |||||||||
|---|---|---|---|---|---|---|---|---|---|
|
بنية الپروتين المرتبط بكريسپر.
| |||||||||
| المعهدات | |||||||||
| الرمز | CRISPR_assoc | ||||||||
| Pfam | PF08798 | ||||||||
| عشيرة Pfam | CL0362 | ||||||||
| InterPro | IPR010179 | ||||||||
| CDD | cd09727 | ||||||||
| |||||||||
| الپروتين كاس 2 المرتبط بكريسپر | |||||||||
|---|---|---|---|---|---|---|---|---|---|
|
البنية البلورية للپروتين المفترض tt1823
| |||||||||
| المعهدات | |||||||||
| الرمز | CRISPR_Cas2 | ||||||||
| Pfam | PF09827 | ||||||||
| InterPro | IPR019199 | ||||||||
| CDD | cd09638 | ||||||||
| |||||||||
| الپروتين Cse1 المرتبط بكريسپر | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| المعهدات | |||||||||
| الرمز | CRISPR_Cse1 | ||||||||
| Pfam | PF09481 | ||||||||
| InterPro | IPR013381 | ||||||||
| CDD | cd09729 | ||||||||
| |||||||||
| الپروتين Cse2 المرتبط بكريسپر | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| المعهدات | |||||||||
| الرمز | CRISPR_Cse2 | ||||||||
| Pfam | PF09485 | ||||||||
| InterPro | IPR013382 | ||||||||
| CDD | cd09670 | ||||||||
| |||||||||
التطور والتوزيع
التطور
A bioinformatic study has suggested that CRISPRs are evolutionarily conserved and cluster into related types. Many show signs of a conserved secondary structure.
CRISPR/Cas can immunize bacteria against certain phages and thus halt transmission. For this reason, Koonin described CRISPR/Cas as a Lamarckian inheritance mechanism. However, this was disputed by a critic who noted, "We should remember [Lamarck] for the good he contributed to science, not for things that resemble his theory only superficially. Indeed, thinking of CRISPR and other phenomena as Lamarckian only obscures the simple and elegant way evolution really works".
التطور المشهجر
Analysis of CRISPR sequences revealed coevolution of host and viral genomes. Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas-mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during interaction with eukaryotic hosts. For example, Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence.
The basic model of CRISPR evolution is newly incorporated spacers driving phages to mutate their genomes to avoid the bacterial immune response, creating diversity in both the phage and host populations. To fight off a phage infection, the sequence of the CRISPR spacer must correspond perfectly to the sequence of the target phage gene. Phages can continue to infect their hosts given point mutations in the spacer. Similar stringency is required in PAM or the bacterial strain remains phage sensitive.
المعدلات
A study of 124 S. thermophilus strains showed that 26% of all spacers were unique and that different CRISPR loci showed different rates of spacer acquisition. Some CRISPR loci evolve more rapidly than others, which allowed the strains' phylogenetic relationships to be determined. A comparative genomic analysis showed that E. coli and S. enterica evolve much more slowly than S. thermophilus. The latter's strains that diverged 250 thousand years ago still contained the same spacer complement.
Metagenomic analysis of two acid mine drainage biofilms showed that one of the analyzed CRISPRs contained extensive deletions and spacer additions versus the other biofilm, suggesting a higher phage activity/prevalence in one community than the other. In the oral cavity, a temporal study determined that 7-22% of spacers were shared over 17 months within an individual while less than 2% were shared across individuals.
From the same environment a single strain was tracked using PCR primers specific to its CRISPR system. Broad-level results of spacer presence/absence showed significant diversity. However, this CRISPR added ثلاثة spacers over 17 months, suggesting that even in an environment with significant CRISPR diversity some loci evolve slowly.
CRISPRs were analysed from the metagenomes produced for the human microbiome project. Although most were body-site specific, some within a body site are widely shared among individuals. One of these loci originated from streptococcal species and contained ~15,000 spacers, 50% of which were unique. Similar to the targeted studies of the oral cavity, some showed little evolution over time.
CRISPR evolution was studied in chemostats using S. thermophilus to directly examine spacer acquisition rates. In one week, S. thermophilus strains acquired up to three spacers when challenged with a single phage. During the same interval the phage developed single nucleotide polymorphisms that became fixed in the population, suggesting that targeting had prevented phage replication absent these mutations.
Another S. thermophilus experiment showed that phages can infect and replicate in hosts that have only one targeting spacer. Yet another showed that sensitive hosts can exist in environments with high phage titres. The chemostat and observational studies suggest many nuances to CRISPR and phage (co)evolution.
التطبيقات
By the end of 2014 some 1000 research papers had been published that mentioned CRISPR. The technology had been used to functionally inactivate genes in human cell lines and cells, to study Candida albicans, to modify yeasts used to make biofuels and to genetically modify crop strains. CRISPR can also be used to change mosquitos so they cannot transmit diseases such as malaria.
CRISPR-based re-evaluations of claims for gene-disease relationships have led to the discovery of potentially important anomalies.
هندسة الجينوم
CRISPR/Cas9 genome editing is carried out with a Type II CRISPR system. When utilized for genome editing, this system includes Cas9, crRNA, tracrRNA along with an optional section of DNA repair template that is utilized in either non-homologous end joining (NHEJ) or homology directed repair (HDR).
المكونات الرئيسية
| المكون | الوظيفة |
|---|---|
| crRNA | Contains the guide RNA that locates the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex. |
| tracrRNA | Binds to crRNA and forms an active complex. |
| sgRNA | Single guide RNAs are a combined RNA consisting of a tracrRNA and at least one crRNA |
| Cas9 | Protein whose active form is able to modify DNA. Many variants exist with differing functions (i.e. single strand nicking, double strand break, DNA binding) due to Cas9's DNA site recognition function. |
| Repair template | DNA that guides the cellular repair process allowing insertion of a specific DNA sequence |
CRISPR/Cas9 often employs a plasmid to transfect the target cells. The main components of this plasmid are displayed in the image and listed in the table. The crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the cell's DNA. The crRNA must bind only where editing is desired. The repair template is designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence.
Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells.
البنية
التسليم
Scientists can use viral or non-viral systems for delivery of the Cas9 and sgRNA into target cells. Electroporation of DNA, RNA or ribonucleocomplexes is the most common and cheapest system. This technique was used to edit CXCR4 and PD-1, knocking in new sequences to replace specific genetic "letters" in these proteins. The group was then able to sort the cells, using cell surface markers, to help identify successfully edited cells. Deep sequencing of a target site confirmed that knock-in genome modifications had occurred with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. However, hard-to-transfect cells (stem cells, neurons, hematopoietic cells, etc.) require more efficient delivery systems such as those based on lentivirus (LVs), adenovirus (AdV) and adeno-associated virus (AAV).
التعديل
CRISPRs have been used to cut five to 62 genes at once: pig cells have been engineered to inactivate all 62 Porcine Endogenous Retroviruses in the pig genome, which eliminated transinfection from the pig to human cells in culture. CRISPR's low cost compared to alternatives is widely seen as revolutionary.
Selective engineered redirection of the CRISPR/Cas system was first demonstrated in 2012 in:
- Immunization of industrially important bacteria, including some used in food production and large-scale fermentation
- Cellular or organism RNA-guided genome engineering. Proof of concept studies demonstrated examples both in vitro and in vivo
- Bacterial strain discrimination by comparison of spacer sequences[]
التعديل المحكوم للجينوم
Several variants of CRISPR/Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules. These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation, or fusing similar light responsive domains with two constructs of split-Cas9, or by incorporating caged unnatural amino acids into Cas9, or by modifying the guide RNAs with photocleavable complements for genome editing.
In 2017, researchers successfully used CRISPR-Cas9 as a treatment in a mouse model of human genetic deafness, by genetically editing the DNA in some cells in the ears of live mice.
Knockdown/التفعيل
Using "dead" versions of Cas9 (dCas9) eliminates CRISPR's DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dCas9s, enabling them to turn almost any gene on or off or adjust its level of activity. Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated, epigenetically modifying the gene. This modification inhibits transcription. Conversely, CRISPR-mediated activation (CRISPRa) promotes gene transcription. Cas9 is an effective way of targeting and silencing specific genes at the DNA level. In bacteria, the presence of Cas9 alone is enough to block transcription. For mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA sequences called promoters that immediately precede the target gene.
Cas9 was used to carry synthetic transcription factors that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter.
تعديل الرنا
In 2016 researchers demonstrated that CRISPR from an ordinary mouth bacterium could be used to edit RNA. The researchers searched databases containing hundreds of millions of genetic sequences for those that resembled Crispr genes. They considered the fusobacteria Leptotrichia shahii. It had a group of genes that resembled CRISPR genes, but with important differences. When the researchers equipped other bacteria with these genes, which they called C2c2, they found that the organisms gained a novel defense.
Many viruses encode their genetic information in RNA rather than DNA that they repurpose to make new viruses. HIV and poliovirus are such viruses. Bacteria with C2c2 make molecules that can dismember RNA, destroying the virus. Tailoring these genes opened any RNA molecule to editing.
نماذج الأمراض
الوظائف
التحرير
Reversible knockdown
التنشيط
الاستخدام حسب العاثية
براءات الاختراع والترويج
المجتمع والثقافة
تعديل خط جنس بشري
At least four labs in the US, labs in China and the UK, and a US biotechnology company called Ovascience announced plans or ongoing research to apply CRISPR to human embryos. Scientists, including a CRISPR co-inventor, urged a worldwide moratorium on applying CRISPR to the human germline, especially for clinical use. They said "scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans" until the full implications "are discussed among scientific and governmental organizations". These scientists support basic research on CRISPR and do not see CRISPR as developed enough for any clinical use in making heritable changes to humans.
عوائق سياسية أمام الهندسة الوراثية
الاعتراف بأهميته
في 2012 و2013، حصلت كرسپر على المركز الثاني في مسابقة مجلة ساينس لجائزة اختراق العام. وفي 2015، فازت بالجائزة. أُدرِجت كرسپر كأحد عشر تكنولوجيات اختراقية في مجلة إم‌آي‌تي تكنولوجي رڤيو في 2014 and 2016. وفي 2016، فازت جنيفر دودنا وإمانوِل شارپنتييه، مع رودلف بارانگووفيليپ هورڤات وفنگ ژانگ بجائزة گيردنر العالمية. وفي 2017، حصلت جنيفر دودنا وإمانوِل شارپنتييه على جائزة اليابان لإختراعهم الثوري CRISPR-Cas9 في طوكيو، اليابان.
قواطع بديلة
CRISPR-DR2: Secondary structure taken from the Rfam database. Family RF01315.
CRISPR-DR3: Secondary structure taken from the Rfam database. Family U1 snRNA.
CRISPR-DR5: Secondary structure taken from the Rfam database. Family RF011318.
CRISPR-DR6: Secondary structure taken from the Rfam database. Family RF01319.
CRISPR-DR8: Secondary structure taken from the Rfam database. Family RF01321.
CRISPR-DR9: Secondary structure taken from the Rfam database. Family RF01322.
CRISPR-DR19: Secondary structure taken from the Rfam database. Family RF01332.
CRISPR-DR41: Secondary structure taken from the Rfam database. Family RF01350.
CRISPR-DR52: Secondary structure taken from the Rfam database. Family RF01365.
CRISPR-DR57: Secondary structure taken from the Rfam database. Family RF01370.
CRISPR-DR65: Secondary structure taken from the Rfam database. Family RF01378.
انظر أيضاً
- تداخل الكريسپر
- باعث وراثي
- Transcription activator-like effector nuclease (TALEN)
- Zinc finger nuclease
- CRISPR/Cas Tools
- Gene knockout
- RNAi
- SiRNA
- Synthetic biology
- Surveyor nuclease assay
- DRACO
المصادر
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