Thanks to Jason Wright for suggesting the Golden Gate Assembly cloning strategy and Katherine Helming and members of Orkin lab, particularly Jian Xu, Guoji Guo, Elenoe Smith, and Partha Das for helpful discussions. This work was supported by NIH R01HL032259 and P30DK049216 (Center of Excellence in Molecular Hematology) to S.H.O. and NIDDK K08DK093705 to D.E.B.

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We have no conflicts of interest related to this report.

CRISPR / מערכת Cas9 ניתן להשתמש כדי ליצור מחיקות הגנומי של מגוון רחב של גדלים. למרות שראינו כי שכיחותה של מחיקה משתנה ביחס הפוך ביחס לגודל מחיקה נועד, הצלחנו להתאושש מחיקות של עד 1 מגה, ומחיקות של עד 100 kb להניב באופן שגרתי שיבוטים biallelic נמחק מרובים. יש לנו לא הבחנתי בהפסד ביעילות של מחיקות ברצף מציגים לקו תא. אסטרטגיה זו יכולה לשמש ליצירת מחיקה קומבינטורית של גנים רבים ואלמנטים. תהליך קבלת שיבוטים מחיקת biallelic יכול להיות מזורז על ידי הערכת המספר המינימאלי של שיבוטים שצריכים לסרוק על בסיס גודל מחיקה כדי לקבל את המספר הרצוי של שיבוטים עם מחיקת biallelic 12. היכולת להשיג מחיקת monoallelic עם היעדר מחיקת biallelic בהפצה הסתברותי יכולה להצביע קטלני תא הקשורים לאובדן תפקוד מלא. fr הנמוך<html> equency או השמטות נעדרו יכול לשקף מספר התרחישים כוללים transfection העני, sgRNAs לא יעיל או לא יעילים פריימרים PCR הקרנה (בשל חוסר שליטה חיובית כדי לאמת פריימרים PCR למסך למחיקה). תאי GFP + יכולים לשמש כתחליף ליעילות transfection (ראה שלב 5.2), כך לירידה בGFP + תאים סביר משקפת transfection העני ותוצאה כך ירדו יעילות מחיקה. שימוש בשני זוגות sgRNA שונים עם פריימרים הקרנה עצמאיים יכול לעזור שליטה לsgRNA לא יעיל והקרנת פריימרים PCR ומקסם את סיכויי קבלת שיבוטים מחיקת biallelic. מיון התא לGFP + תאים מעשיר לאללים מחיקה. בעוד שניתן להשמיט את הצעד הזה, השמטה צפויה מחייבת הקרנה יותר שיבוטים לזהות אלו עם מחיקות monoallelic או biallelic. במידה שיעילות transfection עשויה להיות מותאמת, היינו מצפה יעילות עריכת הגנום להיות משופרת. = &quot;Jove_content&quot; ילדה&gt; אירועי NHEJ העומדים בבסיס מחיקות ותוצאת תיקון מקומי בסדרה של אללים עם מגוון רחב של indels באתרי היעד. התוצאה העיקרית היא ~ 1 קטנה - 10 הוספות נ&quot;ב או יותר נפוצה מחיקות באתר של מחשוף מכוון sgRNA (איור 2). לעתים קרובות אללים אלה מופיעים להיות התוצאה של 34,35 תיקון מבוסס microhomology. יש לציין כי אסטרטגית זיהוי מבוססת PCR אנו מתארים לא תזהה את הוספות גדולות יותר או מורכבות יותר, מחיקות, תנוחות הפוכות, או ארגון מחדש. למרות שהאירועים אלה הם פחות נפוצים, שראינו בו לא שיבוטים מחיקה ולא amplicons הלא המחיקה ניתן הייתה לזהות, ועל חקירה נוספת משקפים תוצאות אלה מורכבות יותר. אנו צפינו בCRISPR הנרחב / התיווך Cas9 &quot;צלקות&quot; של אללים שאינם מחיקה משיבוטי monoallelic ולא מחיקה (ראה איור 2). &quot;צלקות&quot; אלה כוללותindels הקטן המיוצר באתר מחשוף sgRNA ללא מחיקה המיועדת (כלומר, מחיקה של המגזר להתערב בין sgRNAs A ו- B). צלקות אלה לעתים קרובות להפריע הכרת מטרה על ידי sgRNA. לכן היינו ממליץ על זהירות בretargeting אללים בתאים שנחשפו בעבר לsgRNAs תוך שימוש באותה sgRNAs. אסטרטגית מיקוד מחדש מוצלחת יותר היה לנצל sgRNA ייחודי רצפים שונים מהעבר &quot;מצולק&quot; אתרי הכרה. במקרים שבם זוג sgRNAs מכיר רצפי exonic (איור 1, למטה), אללים frameshift עשוי להיות מיוצר גם בהיעדרו של מחיקה. לכן, יכולים להיות מועשרים שיבוטים מחיקת monoallelic לאובדן של פונקציה בשל התדירות הגבוהה של מוטציות frameshift על שאינם נמחק אלל 12. דאגה אחת עם CRISPR / מערכת Cas9 היא השפעות חוץ-היעד, כלומר, שינוי הגנומי באתרים לא רצויים 36-38. Rדיווחי ecent הציעו כי RNAs מדריך קצר עם 17-19 נוקלאוטידים יכול להפחית את התדירות של CRISPR / Cas9 מבוסס השפעות חוץ-יעד 39. בנוסף, אסטרטגיה כפול nicking באמצעות שני מדריכים לכל אתר יעד עם nickase יכולה לשמש כדי ליצור DSBs תוך מזעור השפעות חוץ-יעד 7. לחלופין, מקביל לאסטרטגיות המשמשות לRNAi, אנו מציעים שזוגות שונים של sgRNAs עם רצפי protospacer שאינו חופפים לשמש כדי להוכיח כי הפנוטיפ שנצפה היא התוצאה של CRISPR / השינוי Cas9 על-היעד בניגוד לפוטנציאל מחוץ היעד אפקט. גישה נוחה תהיה לתכנן לפחות שני זוגות sgRNA סמוכים אך אינם חופף, כך שקבוצה אחת של פריימרים הקרנה (ראה שלב 2) יכולה לשמש לזוגות רבים sgRNA (איור 1 א). יתר על כן, משלים קו תא מחיקה על ידי reintroducing הרצף החסר ו / או גן שיבש יכול לבסס קשר סיבתי ביןמחיקה נתון הגנומי ופנוטיפ. לביולוגים עבודה עם מערכות מודל סלולארי, RNAi ייצג כלי רב עוצמה לגנומיקה פונקציונלית. עם זאת, מגבלות של גישה זו כללו הפחתה חלקית ברמות תעתיק mRNA היעד, ההטרוגניות של השפעה של חומרים כימיים עצמאיים מיקוד אותו הגן, וידועות השפעות חוץ-היעד כוללים אפקטים מבוססי זרע ולא זרע 40-42. אסטרטגיות עריכת הגנום מבטיחות לטפל ברבים מחששות אלה ומייצגים גישה מרגשת, משלימה להפרעות גנטיות פוטנציאליים 8,36,37. יתר על כן, עריכת הגנום מאפשרת לחקר ללא קידוד אלמנטים גנטיים בדרך לא ניתן על ידי RNAi ומאתגרת על ידי מיקוד קונבנציונלי גישות 25. אנו מעודדים את הדור של מחיקות הגנומי ידי CRISPR / Cas9 כשיטת חזקה וספציפית כדי לייצר ולאפיין אללים פסד של פונקציה.

Recent advances in genome engineering technology have allowed for unprecedented opportunities for site-specific modification of the genome. This technology may be utilized to investigate the function of genes and regulatory elements via prospective genetic perturbation. Zinc finger nucleases (ZFNs), transcription-activator like (TAL) effector nucleases (TALENs), and CRISPR/Cas9 RNA-guided nucleases each leverage customizable DNA specificity to localize a nuclease for the introduction of DSBs1&#8211;3. The resulting DSBs can be repaired by indel-forming NHEJ or by homology-directed repair (HDR) using a donor template4.
The CRISPR/Cas9 nuclease pathway, an adaptive immune system in prokaryotic cells5, has been recently adapted for mammalian genome engineering2,3. This tool has been demonstrated to be an inexpensive, efficient, and reliable genome engineering technique6. Briefly, a complex of Streptococcus pyogenes-derived Cas9 nuclease and a sgRNA achieve target recognition via Watson-Crick base-pairing with cognate genomic DNA sequences. sgRNAs include 20-mer sequences complementary to genomic sequences adjacent to an obligate protospacer adjacent motif (PAM) NGG. Cas9 induces a DSB at a predictable position within the target site. Additionally, variants of Cas9 with single-strand cleavage capacity or catalytic inactivity may be used to facilitate &#8220;nicking&#8221; or transcriptional regulation respectively7&#8211;9. CRISPR/Cas9 has been used for a wide range of applications including both knock-in and knockout10,11, large-scale genomic deletions12&#8211;14, pooled library screening for gene discovery15,16, genetic engineering of numerous model organisms10,11,17&#8211;21, as well as gene therapy22,23.
Here we describe a protocol for efficient deletion of desired genomic regions. The protocol includes CRISPR design, cloning, and delivery, as well as deletion, identification, and characterization. Genomic deletions can be generated by the introduction of two CRISPR sgRNAs with Cas9 to induce repair of the resultant two DSBs by NHEJ with deletion of the intervening segment. This strategy has been used to create deletions ranging from one kilobase to over one megabase12. Deletions can be informative for the study of genes and other genetic elements, either in isolation or in combination. There are several potential advantages of genomic deletions as compared to HDR or single-site small indel production. First, this method capitalizes on the high efficiency of NHEJ in many cellular contexts7. The high frequency of deletion limits the number of clones needed to be screened to identify informative clones. Deletion frequency is inversely related to deletion size. Biallelic deletion clones may be retrieved at frequencies at least as great as of probabilistic expectation12. Second, both monoallelic and biallelic deletions may be easily identified and distinguished by conventional PCR, simplifying the screening process. Strategies relying on small indels or point mutations may require RFLP, allele-specific PCR, T7EN1 cleavage assay, Sanger sequencing, RT-qPCR, or immunoblotting, which may be more laborious. Third, by removing a substantial portion of a gene or element of interest, a reliable loss-of-function allele may be obtained. In contrast, frameshift mutations in protein-coding sequences may not always induce nonsense-mediated decay, may produce a hypomorphic or neomorphic allele, or target an exon excluded from an alternate isoform24. Finally, deletions may be particularly revealing for the study of non-coding DNA such as regulatory elements since frameshift mutations as produced by single-site indels would not be relevant25.

<table border="0" cellpadding="0" cellspacing="0" width="842">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:388px;">Name of Material/ Equipment</td>
			<td style="width:168px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 Polynucleotide Kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase (with associated ligation buffer)</td>
			<td>New England Biolabs</td>
			<td>M0202T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5&#39;-Triphosphate (ATP)</td>
			<td>New England Biolabs</td>
			<td>P0756S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA, Molecular Biology Grade</td>
			<td>New England Biolabs</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BbsI Restriction Enzyme (with associated NEB Buffer 2.1)</td>
			<td>New England Biolabs</td>
			<td>R0539S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pSpCas9(BB) (pX330)</td>
			<td>Addgene</td>
			<td>42230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S.O.C. Medium</td>
			<td>Life Technologies</td>
			<td>15544-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BTX ECM 830</td>
			<td>Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BTX Solution and 2mm Cuvettes</td>
			<td>Harvard Apparatus</td>
			<td>45-0803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pmaxGFP Plasmid</td>
			<td>Lonza</td>
			<td>VPA-1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickExtract DNA Extraction Solution</td>
			<td>Epicentre</td>
			<td>QE09050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HotStarTaq PCR Master Mix Kit</td>
			<td>Qiagen</td>
			<td>203443</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zero Blunt PCR Cloning Kit</td>
			<td>Life Technologies</td>
			<td>K2700-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of genomic deletions in mammalian cell lines via crispr/cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

מטרת ניסוי זה הייתה המחיקה של Pim1 בתאי MEL. שימוש בזוגות רבים שאינו חופף sgRNA (כלומר, רצפי protospacer עצמאיים) עשויים לעזור לשלוט על השפעות חוץ-יעד (איור 1 א). פנוטיפ עקבי יהיה סביר יותר כתוצאה מהשפעה על היעד בניגוד להשפעה מחוץ יעד משותפת של רצפי protospacer עצמאיים מרובים. כל זוג היה להוביל לייצור של נקודת עצירה מחיקה ייחודית. אם קרוב זה לזה (כלומר, n פחות מ ~ 150 נ&quot;ב), יכולים לשמש באותה פריימרים הקרנה לזהות מחיקות המיוצרות על ידי כל קבוצה של sgRNAs. מחיקות הגנומי עלולות לשבש גנים באמצעות זוגות sgRNA במקומות שונים ביחס לגן (איור 1). לדוגמא, זוג sgRNA יכול לאגף גן למחיקה של גוף הגן כולו; הזוג יכול להיות ממוקם בתוך שני אקסונים, עם הפוטנציאל ליצירת indels frameshift גם אם allel אחד או שניהםes לא נמחקו; או הזוג עשוי לאגף אקסון ספציפי כדי לאפשר שיבוש איזופורם מסוים. אסטרטגית המחיקה משמשת לPim1 הייתה לתכנן איגוף sgRNAs למחוק את כל גוף הגן, מחיקת 8 kb (איור 2). אסטרטגיה זו נבחרה בין שאר בשל גודלו הקטן יחסית של גן Pim1. דוגמא זו מראה PAM אחד (ירוק) בחלק העליון (ווטסון) הגדיל וPAM אחד בתחתית הגדיל (קריק); עם זאת, DSB הוא עצמאי של לוקליזציה רצף PAM לגדיל העליון או תחתון. זוגות sgRNA יכולים לקבל את שניהם רצפי PAM על הגדיל העליון, הן על הגדיל התחתון, או אחד מכל אחד. תוך שימוש בפרוטוקול שתואר לעיל, שתי sgRNA נועדו, משובטים לתוך וקטור ביטוי pX330, ונמסר לתאי MEL על ידי electroporation יחד עם כתב ה- GFP (איור 2 א). 3% העליונים של תאי GFP + מוינו יומיים לאחר electroporation ומצופים clonally בהגבלת דילול. מסךפריימרים ing תוכננו כמתואר בשלב 2 וכפי שמוצג באיור 2. תנאי PCR היו מותאמים באמצעות gDNA מבודד מתאי MEL הורים ומתאים &quot;בתפזורת&quot; מסודרים. 10 ימים לאחר הציפוי, gDNA היה מבודד מכל השיבוטים והוקרן למחיקה באמצעות PCR, שזיהה שאינו מחיקה, monoallelic ושיבוטי מחיקת biallelic פי הדפוסים של אי-מחיקה (ND) ומחיקה (D) amplicons (איור 3) . שיבוטים ללא מחיקה זוהו כבעלי הנוכחות של amplicon שאינו המחיקה והעדר amplicon המחיקה. שיבוטים Monoallelic זוהו כבעלי הנוכחות של שני הלא-המחיקה וamplicons מחיקה. שיבוטים Biallelic זוהו כבעלי העדר amplicon הלא המחיקה ואת הנוכחות של amplicon המחיקה. למחיקה זו, 400 שיבוטים הוקרנו אשר זיהה 126 שיבוטים מחיקת monoallelic ו -32 delet biallelic<html> שיבוטים יון (חשוב לשים לב כי תדירות מחיקה משתנה עם גודל מחיקה 12). שיבוטים מחיקת Biallelic נבחרו ועברו לצנצנות עם 8 מיליליטר של תקשורת. לאחר 5 ימים ומאפשרים להרחבה, כל שיבוט היה הערכה מחדש על ידי PCR של gDNA כדי לאשר amplicons מחיקה ומחיקת biallelic היו נתונים לסנגרו רצף לזהות את המחיקה המדויקת (איור 2). ההטרוגניות בתוך amplicons המחיקה משקפת תיקון NHEJ יוצרי indel מושלם. רצף של אלל הלא המחיקה בשיבוטי מחיקת monoallelelic חשפו indels ברוב המכריע של המקרים, הוכחה כי אפילו אלל-נמחק לא נערך לעתים קרובות על ידי CRISPR / Cas9, אשר עשוי להיות חשוב עבור יישומים בהם שני שיבוטים מחיקת monoallelic וbiallelic נדרשים . RNA היה מבודד משיבוטי מחיקת biallelic ונותח על ידי RT-qPCR כדי לאשר אובדן ביטוי Pim1 (איור 4). דרכים &quot;&gt; <img alt="איור 1" src="/files/ftp_upload/52118/52118fig1highres.jpg" /> איור 1. סכמטי של אסטרטגיות מחיקה אפשריות. () זוגות sgRNA שתי דוגמא למחיקות הגנומי (מוצג בשחור וכתום, בהתאמה). החיצים הכחולים מצביעים על פריימרים לזהות amplicon הלא המחיקה וחצים אדומים מצביעים על פריימרים לזהות amplicon המחיקה. sgRNA ממקם 17 ו -18 מודגשים באדום וכחול בsgRNA-אתרים-1/2 ומודגשים בסגול וכתום בsgRNA-אתרים-B 1 / B 2 עם קו אדום מציין את מחשוף Cas9 חזה בין עמדות 17 ו18. שסעים (B) מכוון CRISPR מוצגים כקווים שחורים אנכיים. החיצים הכחולים מצביעים על פריימרים לזהות amplicon הלא המחיקה וחצים אדומים מצביעים על פריימרים לזהות amplicon המחיקה. <a href="https://www.jove.com/files/ftp_upload/52118/52118fig1large.jpg" target="_blank">אנא לחצו כאן לצפייה</a>גרסה גדולה יותר של דמות זו. <img alt="איור 2" src="/files/ftp_upload/52118/52118fig2highres.jpg" /> איור 2. אסטרטגיה לייצר ולזהות מחיקה של Pim1, כוללים מחיקת רצף ואללים שאינם מחיקות. () סכמטי של אסטרטגית ההקרנה מבוססת PCR לזהות שיבוטים מחיקת Pim1. זוג פריימר אחד ממוקם פנימי למחיקה (חיצים כחולים) וזוג פריימר אחד הוא מקומי חיצוני למחיקה (חצים אדומים). רצפי sgRNA (רצפי protospacer) מוצגים בסגול. הקווים האדומים האנכיים מצביעים על מחשוף Cas9 חזה בין עמדות 17 ו -18 של רצף sgRNA. סנגר רצף (B) מגלה היווצרות indel באתר הכרת sgRNA. רצפי sgRNA מוצגים ברצפים סגולים וPAM בירוק. אירועי מחיקה הם shשלו על ידי מספר דומה של סימני מקף והוספות מודגשים בכחול. קווים אדומים אנכיים מצביעים חזו אתר מחשוף, בין עמדות 17 ו -18 של sgRNA. <a href="https://www.jove.com/files/ftp_upload/52118/52118fig2large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 3" src="/files/ftp_upload/52118/52118fig3highres.jpg" /> איור 3. ג&#39;ל נציג זיהוי שאינו מחיקה, monoallelic, ושיבוטים biallelic. עבור כל שיבוט, הנתיב השמאלי מייצג את amplicon הלא המחיקה (&quot;ND&quot; בכחול) והנתיב הימני מייצג את amplicon המחיקה (&quot;D&quot; באדום ). שיבוטים בודדים מופרדים על ידי קווים מקווקווים. שלושה השיבוטים מחיקת monoallelic הם שיבוטים 5, 6, ו -2 שלושה השיבוטים מחיקת biallelic הם שיבוטים 15, 17, 13. וכפי שניתן לראות בנתונים הרצף, להקת המחיקה לג<html> יש בודד 13 גודל קטן יותר בשל מחיקות גדולות יותר בצומת המחיקה. <img alt="איור 4" src="/files/ftp_upload/52118/52118fig4highres.jpg" /> הפסד איור 4. ביטוי Pim1 בשיבוטי מחיקת biallelic. ביטוי Pim1 חושב לשני שיבוטים מחיקת biallelic על ידי RT-qPCR. הנתונים מנורמלים לGAPDH באמצעות 2 - שיטת ΔCt. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> Protospacer רצף </td></tr><tr><td> sgRNA- </td><td> 5&#39;-TAGGAAAATGACTCGTCACT-3 &quot; </td></tr><tr><td> sgRNA-B </td><td> 5&#39;-GTCTATCCTATCTCGATAAA-3 &quot; </td></tr></tbody></table> טבלה 1. רצפי protospacer 20-mer לשתי sgRNA למחיקה של Pim1. 543 &quot;&gt; <tbody><tr height="23"><td height="23" style="height:23px;width:101px;"></td><td style="width:443px;"> משלים הפוך של רצף Protospacer </td></tr><tr height="23"><td height="23" style="height:23px;"> sgRNA-A-rc </td><td> 5&#39;-AGTGACGAGTCATTTTCCTA-3 &quot; </td></tr><tr height="23"><td height="23" style="height:23px;"> sgRNA-B-rc </td><td> 5&#39;-TTTATCGAGATAGGATAGAC-3 &quot; </td></tr></tbody></table> טבלת 2. השלמה היפוך של רצפי protospacer לsgRNA שתי מטבלת 1. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> רצפים </td></tr><tr><td> sgRNA- </td><td> 5&#39;-CACCTAGGAAAATGACTCGTCACT-3 &quot; </td></tr><tr><td> sgRNA-A-rc </td><td> 5&#39;-AAACAGTGACGAGTCATTTTCCTA-3 &quot; </td></tr><tr><td> sgRNA-B </td><td> 5&#39;-CACCGTCTATCCTATCTCGATAAA-3 &quot; </td></tr><tr><td> sgRNA-B-rc </td><td> 5&#39;-AAACTTTATCGAGATAGGATAGAC-3 &quot; </td></tr></tbody></table> טבלת 3. רצפי Protospacer וההפוך שלהם משלים עם &quot;CACC&quot; ו- &quot;AAAC&quot;, הוסיפו לשיבוט לתוך וקטור pX330 באמצעות אנזים הגבלת BbsI. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> רצפים </td></tr><tr><td> sgRNA- </td><td> 5&#39;- CACCGTAGGAAAATGACTCGTCACT-3 &quot; </td></tr><tr><td> sgRNA-A-rc </td><td> 5&#39;- AAACAGTGACGAGTCATTTTCCTAC-3 &quot; </td></tr><tr><td> sgRNA-B </td><td> 5&#39;- CACCGTCTATCCTATCTCGATAAA-3 &quot; </td></tr><tr><td> sgRNA-B-rc </td><td> 5&#39;- AAACTTTATCGAGATAGGATAGAC-3 &quot; </td></tr></tbody></table> שולחן ביטוי 4. sgRNA מאמרגן U6 של וקטור pX330 מוגבר על ידי התוספת של נוקלאוטיד G לאחר רצף CACC ולפני20-mer. התוספת של נוקלאוטיד G נוסף דורשת תוספת של נוקלאוטיד C בסוף &#39;3 ​​של אוליגו המשלים ההפוך (למשל, sgRNA-A). עם זאת, אם המיקום הראשון של 20-mer (רצף protospacer) הוא כבר נוקלאוטיד G, אין צורך להוסיף G אחר (לדוגמא, sgRNA-B) ואין צורך להוסיף ג &#39;לעמדתו הסופית של ההשלמה ההפוכה אוליגו.

Watch this Scientific Journal Video about Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 at JoVE.com

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

דור של מחיקות הגנומי בשורות תאים יונקים באמצעות CRISPR / Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

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94.9K Views.  Harvard Medical School. CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

Video: Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

המשימה esiRNA עבור הקרנת RNAi בתאי יונקים

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

תהליך המפלצת הירוקה לדור של זני שמרי נשיאת מחיקות Gene מרובות

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

דור של שורות תאים אנושיות יציבות עם טטרציקלין-מושרה (ט ב) shRNA או ביטוי cDNA

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

צמיחה אנאירובית ותחזוקה של שורות תאים בתרבית

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

השתקה הניצוץ: CRISPR/Cas9 הגנום עריכה בדגים חשמליים חלש

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

הנדסת גנום של תאי B אנושיים ראשוניים באמצעות CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&amp;#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

אנדותל בתיווך TGF-β למעבר מסנצ'ימלי (EndMT) וההערכה הפונקציונלית של אפקטי EndMT באמצעות עריכת גנים CRISPR/Cas9

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

בניית מוטנטים הומוזיגוטיים של ארבה נודד בטכנולוגיית CRISPR/Cas9

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

עריכת גנים CRISPR/Cas9 של תאי גזע ותאי אב המטופויאטיים ליישומי ריפוי גנטי

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

CRISPR-Cas9-עריכות מדויקות בתיווך בלבבות דגי זברה

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...