Name: crispr-cas9 gene editing in action
Uploaded: 2019-03-11T09:21:22.000Z
Duration: 1 min 59 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

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Biology

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Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.3

CRISPR

מערכת CRISPR-Cas9 היא כלי עריכת דנ"א ששמו המלא:חזרות פלינדרומיות קצרות מקובצות במרווחים סדורים, וחלבון 9 מקושר קריספר. לאחר ש‫נצפתה לראשונה בחיידקים, CRISPR-Cas9 היא אמצעי הגנה מפני וירוסים. כשדנ"א ויראלי זר נכנס אל החיידק, הוא מעובד למקטעים קטנים יותר, שניתן להכניסם לאזור ב‫גנום החיידק שנקרא לוקוס קריספר.כאשר האזור משועתק, התוצר מקושר עם מולקולות רנ"א קטנות יותר, הנקראות tracrRNAs. הן עשויות לעזור לכוון גם את חלבון ה-Cas9 וגם את הרנא"ז למולקולה. שם הרנ"אז מבקע את התעתיק, ו‫התוצאה הסופית היא מספר תצמידים.כל אחד מהם מורכב מחלבון Cas9, tracrRNA ו-crRNA, ש‫נגזרו מדנ"א שבלוקוס. במבנים אלה מזהה crRNA ומדריך Cas9 לדנ"א ויראלי. שבתורו מבוקע ומושמד.מדענים רותמים CRISPR-Cas9 על ידי סינתזת מולקולות רנ"א בודדות, המחקות tracrRNA ו-crRNA. שיכולות לכוון אל גן המטרה. לדוגמה, כששני רנ"א מדריכים אלה Cas9 מוכנסים לתוך תאים יחד עם, ושניהם מכוונים לאותו הגן, רצף ניתן לחיתוך.לאחר שאזור מטרה זה מוסר, קצוות חתוכים מתחברים מחדש, ונצפות ההשפעות על התאים. לפיכך, מערכת CRISPR-Cas9 היא מנגנון חיידקי שעבר שינוי, וניתנת לגיוס עבור מערך של טכניקות עריכה גנטית.

Thurtle‐Schmidt, Deborah M., and Te-Wen Lo. "ביולוגיה מולקולרית בחוד החנית: סקירה על עריכת גנים CRISPR/CAS9 לסטודנטים לתואר ראשון." חינוך לביוכימיה וביולוגיה מולקולרית 46, מס' 2 (2018): 195–205. [<a href="https://doi.org/10.1002/bmb.21108" target="__blank">Source</a>]

לנדר, אריק ס. "גיבורי הקריספר". תא 164, מס' 1 (14 בינואר 2016): 18–28. [<a href="https://doi.org/10.1016/j.cell.2015.12.041" target="__blank">מקור</a>]

קריספר

<div style="direction:rtl">טכנולוגיות עריכת הגנום מאפשרות למדענים לשנות את ה-DNA של האורגניזם באמצעות הוספה, הסרה או סידור מחדש של חומר גנטי במיקומים גנומיים ספציפיים. טכניקות מסוג זה עשויות לשמש לריפוי הפרעות גנטיות כגון המופיליה ואנמיה חרמשית. כלי מחקר פופולרי ונפוץ לעריכת DNA שיכול להוביל לריפוי בטוח ויעיל להפרעות גנטיות הוא מערכת קריספר (באנגלית: CRISPR, ראשי תיבות של Clustered Regularly Interspaced Short Palindromic Repeats‏) מערכת CRISPR-Cas9 בסיסית מורכבת מאנדונוקלאז של Cas9 ו-RNA קטן שמנחה את Cas9 ל-DNA היעד. <h4>מקור</h4> רצפי קריספר נצפו תחילה בחיידקים ולאחר מכן זוהו בארכאות. חוקרים גילו שמערכת CRISPR-Cas9 משרתת הגנה חיסונית אדפטיבית מפני וירוסים פולשים. חיידקים רבים ורוב הארכאות לוכדים רצפים קצרים של ה-DNA הנגיפי כדי ליצור ספרייה של מקטעי DNA של וירוסים, או מערכי CRISPR. כאשר הפרוקריוטים נחשפים מחדש לאותו נגיף או מחלקה של וירוסים, משתמשים במערכי CRISPR לתמלול מקטעי RNA קטנים שעוזרים לזהות פולשים ויראליים ובהמשך להשמיד DNA ויראלי עם Cas9 או אנדונוקלאז דומה. <h4>שימוש בטכנולוגיית CRISPR-Cas9</h4> CRISPR-Cas9 משמש בדרך כלל במעבדה להסרת DNA ולהכנסת רצף DNA חדש במקומו. כדי להשיג זאת, על החוקרים ליצור תחילה שבר קטן של RNA הנקרא RNA המדריך, עם רצף קצר הנקרא single guide RNA אשר נקשר לרצף מטרה ספציפי ב-DNA גנומי. ה-RNA המדריך יכול גם לקשר עם Cas9 (או אנדונוקליזות אחרות כמו Cpf1). single guide RNA וחלבון Cas9 מנוהלים לתא מעניין שבו ה- RNA המדריך מזהה את רצף ה-DNA של המטרה ו-Cas9 מבקע אותו. מנגנון התא מתקן את הגדילים השבורים על ידי הכנסת או מחיקה של נוקלאוטידים אקראיים, מה שהופך את גן המטרה ללא פעיל. לחלופין, ניתן להכניס לתא רצף DNA מותאם אישית יחד עם RNA המדריך ו-Cas9, המשמש כתבנית למכונות התיקון ומחליף את הרצף שנכרת. זוהי דרך יעילה ביותר עבור חוקרים לבצע 'נוק-אאוט' של גן כדי לחקור את השפעותיו או להחליף גן שעבר מוטציה בעותק רגיל בתקווה לרפא מחלה. <h4>שיקולי אתיקה והיתכנות בבני אדם</h4> כתוצאה מיכולות שינוי הגנים המשמעותיות של מערכת CRISPR-Cas9, היה ויכוח רב על השימוש בה, במיוחד בכל הקשור לעריכת עוברים. מדען סיני טען לאחרונה שיצר תינוקות שנעשו בגנום באמצעות טכנולוגיית קריספר כדי להשבית גן המעורב בזיהום ב-HIV. זה הוביל לזעקה עולמית מצד מדענים המודאגים מהשיקולים האתיים והבטיחותיים של ההליך. רבים כינו את המהלך מוקדם, ואחרים הביעו דאגה לגבי השפעות גנומיות מחוץ למטרה. בעוד שמספר יישומי הביוטק האפשריים עבור מערכת CRISPR-Cas9 הוא רב, חשוב לקחת בחשבון אתגרים עתידיים שעלולים להתעורר כתוצאה מהשימוש בה.</div>

טכנולוגיות עריכת גנום מאפשרות למדענים לשנות את ה- DNA של אורגניזם באמצעות הוספה, הסרה או סידור מחדש של חומר גנטי ב ...

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Genetics

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KEY TERMS AND CONCEPTS

מונחים ומושגים מרכזיים

crispr-cas9 gene editing in action

CRISPR-Cas9 is a genome editing tool that lets scientists change DNA at a specific location. It can add, remove, or rearrange genetic material. Researchers study it because it may help treat genetic disorders such as hemophilia and sickle cell anemia.

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and Cas9 is a CRISPR-associated protein 9. The basic system includes a Cas9 endonuclease, which is an enzyme that cuts DNA, and a small RNA that guides Cas9 to the target sequence. Together, they make CRISPR-Cas9 a precise research tool for DNA editing.

CRISPR sequences were first seen in bacteria and later found in archaea. Scientists discovered that this system acts like an adaptive immune defense against viruses. Many bacteria and most archaea store short pieces of viral DNA in CRISPR arrays. When the same virus returns, these RNA segments help identify it, and Cas9 or a similar endonuclease destroys the viral DNA.

In the lab, CRISPR-Cas9 is often used to remove DNA and replace it with a new sequence. Researchers first design a guide RNA, which contains a guide sequence that matches a target sequence on genomic DNA. The guide RNA and Cas9 are then placed in the cell of interest. The guide RNA finds the target, Cas9 cuts the DNA, and the cell repairs the break by adding or deleting random nucleotides, which can turn the gene off.

Scientists can also add a custom DNA template with the guide RNA and Cas9. The repair machinery can use that template to replace the removed sequence. This makes CRISPR useful for gene knockouts and for replacing a mutated gene with a normal copy in hopes of treating disease. Other endonucleases, such as Cpf1, can also work with guide RNA.

CRISPR-Cas9 has also raised major ethical and safety questions, especially about embryo editing. A Chinese scientist claimed to have used CRISPR to create genome-edited babies by disabling a gene linked to HIV infection. That claim led to global concern because many scientists thought the work was premature and worried about off-target genomic effects. CRISPR has many possible biotech uses, but future challenges still need careful attention.

CRISPR-Cas9 Gene Editing in Action

what is genetic engineering?

How Scientists Edit DNA for New Traits

Genetic engineering changes an organism’s DNA to add useful traits. Scientists use it in bacteria, plants, animals, and even viruses for research, medicine, agriculture, and industry. It can bring major benefits, but it also raises ethical concerns about humans and the food supply. This work is possible because DNA has the same basic structure and genetic code across life. That shared code lets scientists modify DNA in several ways. One method is CRISPR/Cas9, often called molecular scissors,...

dna isolation

Steps in DNA Extraction

DNA extraction is the process of removing and purifying DNA from cells. It is used in many biotechnology and research applications, including molecular cloning. The main goal is to free DNA from the cell and prepare it for later study or use. The first step is cell lysis, which means breaking cells open. Researchers may use a homogenizer, sonicator, or bead beater to physically lyse the cells. They also often add detergent to chemically disrupt the lipid-based cell membranes. This helps...

recombinant dna technology

Recombinant DNA in Cloning and GMOs

Recombinant DNA is made by joining DNA from different sources in the lab. Scientists often use it to clone genes and study them in cells that are easy to handle, such as bacteria. Organisms that carry recombinant DNA are called genetically modified organisms, or GMOs. This technology gives scientists new tools for research, medicine, and agriculture. To build recombinant DNA, researchers place a gene of interest into a vector, which is a DNA carrier that moves foreign DNA into a host cell. The...

PCR Cycle for DNA Amplification

PCR is a laboratory method for making many copies of a DNA segment. It can turn a small amount of DNA into millions or even billions of copies in just a few hours. The process uses a heat-resistant DNA polymerase inside a machine called a thermocycler. PCR was developed by Kary Mullis in 1983, and he received the 1993 Nobel Prize in Chemistry for it. The method is fast, relatively inexpensive, and precise. It has become useful for molecular cloning, gene mutagenesis, pathogen detection, gene...

complementary dna

From mRNA to cDNA for Gene Studies

Complementary DNA, or cDNA, is made from messenger RNA (mRNA) so scientists can study which genes are active in cells and tissues. Only genes that are transcribed into mRNA are expressed. By extracting mRNA from cells, researchers can compare gene expression across samples. cDNA is produced by reverse transcription. This process copies mRNA into DNA, which is useful because mRNA does not contain introns, or non-coding regions, and other regulatory sequences. As a result, cDNA can help...

antibiotic selection

Antibiotic Resistance in Cloning

Antibiotic resistance genes help scientists find bacteria that took up a plasmid with a gene of interest. In genetic engineering, this resistance is used as a marker, not just as a medical concern. The same process also connects to the growing problem of resistant bacteria in the real world. Antibiotics, such as penicillin, stop bacterial growth or kill bacteria. When bacteria naturally have, or are engineered to carry, an antibiotic resistance gene, the antibiotic no longer works on them.

transgenic organisms

Creating Transgenic Plants and Animals

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in-vitro mutagenesis (gene knockouts)

Knockout Mice and Gene Replacement

Knockout mice help researchers study gene function by showing what happens when a gene is turned off. These engineered animals are also useful for modeling human diseases such as cancer, Parkinson’s disease, and diabetes. A specific gene can be targeted by using a targeting vector, which is a piece of engineered DNA that replaces the normal gene and inactivates it. The vector has matching DNA sequences on both ends. These homologous sequences let the vector swap in for the target gene through...

crispr

CRISPR-Cas9 is a genome editing tool that lets scientists change DNA at a specific location. It can add, remove, or rearrange genetic material. Researchers study it because it may help treat genetic disorders such as hemophilia and sickle cell anemia. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and Cas9 is a CRISPR-associated protein 9. The basic system includes a Cas9 endonuclease, which is an enzyme that cuts DNA, and a small RNA that guides Cas9 to the...

gene therapy

Gene Delivery for Serious Diseases

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embryonic stem cells

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Embryonic stem cells are pluripotent cells that have not yet specialized. Pluripotent means they can give rise to many different cell types in the body. Because of this, they are useful in science and medicine for making cells needed in research or for replacing cells lost to damage or disease. These cells come from the inner cell mass of a blastocyst, which is an early embryo about 3 to 5 days after fertilization in humans. At this stage, the embryo has not yet implanted in the uterus. Human...

adult stem cells

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Adult stem cells help maintain tissues by replacing old or damaged cells. These undifferentiated cells divide to make more stem cells or progenitor cells, which then become mature, specialized cells. Small groups of adult stem cells are found in many tissues, including the bone marrow, brain, skin, and gut. The small intestine shows this process clearly. Its lining, called the epithelium, is renewed continuously by adult stem cells. It is also the fastest replaced tissue in the human body,...

induced pluripotent stem cells

Reprogramming Mature Cells into Stem Cells

Reprogramming mature cells into stem cells lets scientists turn specialized cells back into an unspecialized state. These cells are called induced pluripotent stem cells, or iPSCs. Like embryonic stem cells, iPSCs are pluripotent, which means they can form all cell types. To make iPSCs, researchers start with mature cells such as skin fibroblasts or blood cells. The cells are grown in culture, and genes for several transcription factors are delivered with a viral vector. The cells then make...

reproductive cloning

Cloning Animals with SCNT

Reproductive cloning can produce a genetically identical copy of an entire organism. In adult animals, this is usually done with somatic cell nuclear transfer, or SCNT. Another way clones can form is by splitting an early embryo, which is similar to how identical twins arise naturally. In SCNT, an egg cell is taken from an animal and its nucleus is removed. This creates an enucleated egg. A somatic cell, which is any body cell that is not a sex cell, is then taken from the animal to be cloned.

genomics

Genomics and DNA Sequencing

Genomics studies the full set of genetic material in an organism. In humans, that genome includes the DNA carried in 23 pairs of chromosomes in the nucleus, along with mitochondrial DNA. Genomics looks at both coding DNA and non-coding DNA, which helps scientists study how living things are built and how they differ. This field is useful in many areas of biology and medicine. Scientists use genomics to build phylogenetic trees, improve crop productivity and sustainability, help with criminal...

key terms and concepts

Learn how CRISPR-Cas9 edits DNA, uses guide RNA and Cas9, and why scientists debate its use in embryos and gene therapy.

CRISPR-Cas9 Gene Editing in Action | JoVE K12

JoVE Core series brings biology to life through over 300 concise and easy-to-understand animated video lessons that explain key concepts in biology, plus more than 150 scientist-in-action videos that show actual research experiments conducted in today’s laboratories.

biology

myJoVE Corporation

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bacterial-transformation-the-heat-shock-method

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Obtain, evaluate, and communicate information on how molecular biotechnology may be used in a variety of fields.
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BIO1.ETS2.2

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Analyze scientific and ethical arguments to support the pros and cons of applications of a specific biotechnology technique such as stem cell usage, in vitro fertilization, or genetically modified organisms.
EXPLANATION: The utilization of new technologies in any field of science is dependent on both economic and social factors. In addition to evaluating these factors, scientists must also consider long-term consequences that may not be initially apparent. The emphasis should be on the construction of a rationale argument that supports a position on the use of an application with ethical and social impact. Students should begin to appreciate the differences in ethical values that exist, and recognize that discussion of these values is imperative as knowledge and technology continue to advance, even when resolutions of differences can be rare. After investigation of a specific biotechnology application, students can write a position paper and/or participate in a classroom debate.

CRISPR-Cas9 Gene Editing in Action

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