theCrux
theCrux
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Prime editing (pegRNA + RT + nCas9)
References/Resources: www.patreon.com/the_Crux
All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html
What is Prime Editing? How is Base editing done using nCas9? What is pegRNA? What is the structure and design of pegRNA? How is prime editing achieved? More specifically, what is the functional mechanism for prime editing? What is the function of reverse transcriptase in prime editing? In this video, I discuss the development and details of prime editing. It involves a three-component system containing nCas9, pegRNA, and a reverse transcriptase. The video dives into details about prime editing mechanism by highlighting key steps involving pegRNA (primer binding site pairing and reverse transcription of the donor RT template). Prime editing can be used for a variety of applications for short-range genomic editing but it does have its limitations too.
00:00 Why prime editing?
01:55 Features of Prime editing
03:49 Prime editing Mechanism
08:02 Efficiency and Prime editing
CRISPR-Cas9 in genome editing: ua-cam.com/video/7ESZTE6rjLI/v-deo.html
CRISPRi/a (dCas9): ua-cam.com/video/bRlQcIAtThk/v-deo.html
Base editing: ua-cam.com/video/B1Bv-UmxugM/v-deo.html
References/Resources: www.patreon.com/the_Crux
*Must watch playlists*
All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html
All videos on DNA Replication: ua-cam.com/play/PL0Ymnd-zt4IgLefDaeqicJbCBnbLqnDJ7.html
All videos on Transcription: ua-cam.com/play/PL0Ymnd-zt4Ij2VcAOHNUweftSElVsc-MX.html
All videos on Translation: ua-cam.com/play/PL0Ymnd-zt4IjAAAMlPLDIXbs692Yp14xa.html
Consider supporting the growth of this channel by becoming a Patron: www.patreon.com/the_Crux
I appreciate your support :)
Переглядів: 170

Відео

Base editing with CRISPR-Cas9
Переглядів 258День тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html What is Base editing? What is nCas9? How is Base editing done using CRISPR Cas9? What are the types of base editing methods? How is deamination involved in base editing? How can base editing be used? What are the limitations of base editing? In this video, I ...
dCas9: CRISPRi CRISPRa CRISPR-ON CRISPR-OFF (Beyond Cas9 Genome Editing)
Переглядів 1,1 тис.Місяць тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html What is CRISPRi (CRISPR interference)? What is CRISPRa (CRISPR activation)? What is a dCas9 and how to use dCas9 for CRISPRi and CRISPRa experiments? dCas9 can be fused with effector domains to regulate gene expression for functional studies. In addition, you...
CRISPR KO vs CRISPR KI experiment using CRISPR-Cas9
Переглядів 7442 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html How to design a CRISPR experiment? How to perform surveyor assay to check gRNA targeting efficiency? How to perform a CRISPR knock-out experiment? How to perform a CRISPR knock-in experiment? What is the difference between the steps to perform a crispr deleti...
How to design and express CRISPR guide RNA [pX330 plasmid, Tutorial]
Переглядів 1,2 тис.2 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html What is a typical CRISPR Cas9 protocol? What is the workflow of a typical CRISPR protocol? How to design CRISPR Cas9 guide RNA for a CRISPR experiment and clone crispr guides in a plasmid (pX330)? In this video, I discuss a typical CRISPR protocol workflow an...
Genome Editing with CRISPR-Cas9
Переглядів 7 тис.3 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html What is CRISPR-Cas9 technology? How does CRISPR-Cas9 genome editing work? What is the mechanism of genome engineering with CRISPR-Cas9? What is a PAM sequence? Where is the PAM sequence location and what is its function? What is the protospacer adjacent motif...
CRISPR-Cas9-mediated Adaptive Immunity Mechanism in Bacteria [3 Steps of CRISPR-Cas system]
Переглядів 2,1 тис.3 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html What is CRISPR-Cas9? How does CRISPR-Cas9 work in its natural context? How does CRISPR-Cas9 protect bacteria against pathogens by providing adaptive immunity? What are the different types of CRISPR systems? CRISPR-Cas9 is a Type II-A CRISPR system. In general...
A Brief History of Genome Editing
Переглядів 6333 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html This video briefly discusses the history of genome editing and how historical events have led to the current state of the art. The video discusses parallel timelines that have interwoven to result in modern methods today. It all of course rests on the discove...
Protein Expression Vectors - tet-OFF and tet-ON (tTA/rtTA + Tet based gene regulation) [Part 5]
Переглядів 1,2 тис.3 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html What are the types of tet expression systems? What is a tet-ON and tet-OFF expression systems? How do tetracycline inducible expression systems work? This video discusses the regulation of protein expression using a tetracycline inducible or repressible based...
Protein Expression Vectors (pET vector) - Induction of Protein Expression (IPTG + T7 Pol) [Part 4]
Переглядів 2 тис.4 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html What is a pET vector? What is a pET expression system? How do pET vector expression systems work? What are inducible expression systems and how do pET vector systems work? What is the function of T7 RNA polymerase in pET vectors and how does T7 RNA polymerase...
Protein Expression Vectors - Examples (Fusion Proteins, Insulin, and Cas9) [Part 3]
Переглядів 7034 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html In this video, we explore some common protein expression vector designs and how they are constructed. These are helpful examples and applications of protein expression vectors to intuitively understand the protein expression vector construction. We discuss 5 ...
Protein Expression Vectors - Template, Codon Bias, Affinity Tags and Epitopes [Part 2]
Переглядів 6264 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html In this video, we explore how to obtain templates for protein expression vectors depending on the gene structure (intron and exon language). Prokaryotic genes are intron-less, whereas eukaryotic genes often have introns. Obtaining template for protein express...
Protein Expression Vectors - Expression Host and Recombinant Proteins [Part 1]
Переглядів 1,1 тис.4 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html Protein Expression vectors are used to make protein from the template DNA. In this video, we discuss some of the common applications of protein expression vectors and the complications involved with protein expression when changing the host i.e. heterologous ...
RNA Expression Vectors - Design, Applications, and Examples (In vitro Transcription)
Переглядів 5554 місяці тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html RNA Expression vectors (different from protein expression vectors) are used for the sole purpose of making RNA from a template DNA. In this video, we discuss some of the common applications of RNA expression vectors and their rules/principles of design. Final...
Golden Gate Cloning or assembly - Type IIS (BsaI) restriction enzyme based cloning
Переглядів 3,3 тис.7 місяців тому
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ua-cam.com/play/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf.html Golden Gate Cloning or Golden gate assembly is a restriction enzyme based cloning that uses type IIS restriction enzymes. It is a powerful way to clone large numbers of inserts in a single reaction without the need of using multiple enzymes. This video discus...
Gateway Cloning: Simple and Multisite Gateway Cloning - LR and BP Cloning
Переглядів 2,9 тис.9 місяців тому
Gateway Cloning: Simple and Multisite Gateway Cloning - LR and BP Cloning
TOPO Cloning - TOPO-Blunt, TOPO-TA, TOPO-directional
Переглядів 5 тис.Рік тому
TOPO Cloning - TOPO-Blunt, TOPO-TA, TOPO-directional
Cloning with Restriction Enzymes (5 Levels) - Traditional Molecular Cloning
Переглядів 1,8 тис.Рік тому
Cloning with Restriction Enzymes (5 Levels) - Traditional Molecular Cloning
Plant cloning vectors - Disarmed Ti plasmid, Cointegrate vectors, Binary vectors - T DNA transfer
Переглядів 6 тис.Рік тому
Plant cloning vectors - Disarmed Ti plasmid, Cointegrate vectors, Binary vectors - T DNA transfer
Yeast cloning vectors - YIp, YEp, YRp, YCp, YACs - Shuttle plasmid vectors
Переглядів 7 тис.Рік тому
Yeast cloning vectors - YIp, YEp, YRp, YCp, YACs - Shuttle plasmid vectors
P1 Phage and PAC cloning vector - Pac sites, Cre-loxP, and SacB lethality
Переглядів 2,3 тис.Рік тому
P1 Phage and PAC cloning vector - Pac sites, Cre-loxP, and SacB lethality
F plasmid, BACs, Fosmid cloning vector - Fosmids are small BACs (Bacterial Artificial Chromosomes)
Переглядів 1,8 тис.Рік тому
F plasmid, BACs, Fosmid cloning vector - Fosmids are small BACs (Bacterial Artificial Chromosomes)
Lambda Phage vector, Cosmid cloning vector - spi phenotype and in vitro packaged lambda virus
Переглядів 8 тис.Рік тому
Lambda Phage vector, Cosmid cloning vector - spi phenotype and in vitro packaged lambda virus
Plasmid and Phagemid cloning vector - Selectable vs. Screening marker
Переглядів 4,7 тис.Рік тому
Plasmid and Phagemid cloning vector - Selectable vs. Screening marker
Dam and Dcm methylation (restriction independent methyl-transferases) affects restriction enzymes
Переглядів 2,1 тис.Рік тому
Dam and Dcm methylation (restriction independent methyl-transferases) affects restriction enzymes
Restriction Enzymes (Endonucleases) in Molecular Cloning
Переглядів 2,2 тис.Рік тому
Restriction Enzymes (Endonucleases) in Molecular Cloning
Genetic Engineering - Classic vs Modern methods
Переглядів 4,1 тис.Рік тому
Genetic Engineering - Classic vs Modern methods
RNA Polymerase III Transcription - Promoters, TFs, Initiation, Elongation, and Termination steps
Переглядів 4,6 тис.Рік тому
RNA Polymerase III Transcription - Promoters, TFs, Initiation, Elongation, and Termination steps
Prokaryotic Transcription Factors in Elongation and Termination (GreA, GreB, Mfd, NusA)
Переглядів 2,8 тис.Рік тому
Prokaryotic Transcription Factors in Elongation and Termination (GreA, GreB, Mfd, NusA)
Rolling Circle DNA Replication and Amplification - Plasmids and Bacteriophages (M13 + PhiX174)
Переглядів 13 тис.Рік тому
Rolling Circle DNA Replication and Amplification - Plasmids and Bacteriophages (M13 PhiX174)

КОМЕНТАРІ

  • @sheraznazir4559
    @sheraznazir4559 11 годин тому

    great lectures

  • @theCrux
    @theCrux 16 годин тому

    CRISPR-Cas9 in Genome Editing: ua-cam.com/video/7ESZTE6rjLI/v-deo.html CRISPR-KI vs CRISPR-KO: ua-cam.com/video/f0Q6JKu8Xjk/v-deo.html CRISPRi vs CRISPRa: ua-cam.com/video/bRlQcIAtThk/v-deo.html Base Editing: ua-cam.com/video/B1Bv-UmxugM/v-deo.html

  • @gabrielfrank5142
    @gabrielfrank5142 19 годин тому

    Thanks!

  • @danyrivera2492
    @danyrivera2492 День тому

    Hey!! Thank you so much for your videos they have really helped me, would you mind answering what would happen if the desired point of insertion fell in the sgRNA sequence region... I ideally thought the donor DNA would not be affected because the HDR happens after the cut... I would really appreciate your help :)

    • @theCrux
      @theCrux День тому

      Glad the content is useful. If the insertion point overlaps the gRNA sequence then it means that post HDR, at the least, the gRNA target sequence will be split (i.e. you inserted something in it). Therefore, donor will be unaffected and you can even get away without mutating PAM region in the donor. Ideally, the insertion point should overlap the seed region of gRNA (i.e. HDR outcome will split the seed region). If it falls outside of the seed region (for instance, say 3 nts from the 5' end of the gRNA), you will likely find that Cas9 can still target the donor DNA. The farther away the overlap of insertion point from the seed region, the more chances that donor DNA is cut. There are many nuances and caveats to all this, but I hope this helps to guide your thinking.

  • @nopenope6909
    @nopenope6909 2 дні тому

    Great video! Thank you very much!

  • @geofix_123
    @geofix_123 2 дні тому

    Legitimately goated, thanku

  • @Abhinva-w9y
    @Abhinva-w9y 3 дні тому

    how easily you explained the cosmid mechanism....amazing!!

  • @Noname-vz6ky
    @Noname-vz6ky 5 днів тому

    Thank u so much this helps a lot and sooo easy to understand.

  • @theCrux
    @theCrux 8 днів тому

    CRISPR-Cas9 in Genome Editing: ua-cam.com/video/7ESZTE6rjLI/v-deo.html CRISPR-KI vs CRISPR-KO: ua-cam.com/video/f0Q6JKu8Xjk/v-deo.html CRISPRi vs CRISPRa: ua-cam.com/video/bRlQcIAtThk/v-deo.html

  • @Siddiqatif
    @Siddiqatif 9 днів тому

    you're handwriting is immaculate!! A dropdead gorgeous video!! Ok, I got a bit carried away😛

  • @raaw_b_l_a_c_k
    @raaw_b_l_a_c_k 10 днів тому

    Very informative video man thanks for your valuable insights.

  • @RaniaRika-fq5bt
    @RaniaRika-fq5bt 10 днів тому

    Thank you, I couldn't find even half of these explanations anywhere even in advanced lectures from our professors I truly appreciate it

  • @gcfournier3386
    @gcfournier3386 14 днів тому

    Thanks

  • @thurayae
    @thurayae 15 днів тому

    Thanks so much for your amazing videos. I have a question please since it's my first time to learn about crispr. We learn here how to design a crispsr RNA, so wt about the connecting loop and the tracRNA? how does it work without it?

    • @theCrux
      @theCrux 15 днів тому

      Connecting loop between spacer and tracrRNA is not required. Under natural conditions, there is no connecting loop (See: ua-cam.com/video/I0aWfgvpCyU/v-deo.html). The connecting loop is common for plasmid based sgRNA expression because without a connecting loop between tracrRNA and spacer, in vivo it would be inefficient to form a 3 part (Cas9, spacer, tracrRNA) complex for genome editing purposes.

    • @thurayae
      @thurayae 14 днів тому

      ​@@theCrux Thanks so much for your reply. I just watched the video you recommended and really like it. My question was in another case than chosing bacteria for crispr I have watched on another UA-cam video channel. If we chose Electroporation of Cas9 & synthetic gRNA/Trac RNA. To order gRNA, there are many tools that help us to chose the sequence for it like chop chop as you mentioned in one of your videos. What about the tracRNA and the loop in this case?

    • @theCrux
      @theCrux 13 днів тому

      Typically, you never have to worry about connecting loop. In electroporation with pure RNA guides (not plasmid), there are two common ways: (1) either you have full sgRNA ordered (spacer + tracrRNA), or (2) you have the sgRNA split into spacer and tracrRNA (from which you assemble the sgRNA by yourself). For full sgRNA, the connecting loop is part of the design by default provided by the company and according to your selection of Cas enzyme. For two part system, you order the spacer and depending on your Cas selection tracrRNA will have all the features neccessary for complex formation. The respective company will (or should) artificially add an overhang to the spacer that is complementary to the tracrRNA provided, so that a complex can form. In split sgRNA versions, there is no connecting loop (because "split"). In case of plasmid based expression (pX330 etc.), the sgRNA scaffold contains the connecting loop by default, so you don't have to worry about adding one yourself.

  • @mytumin
    @mytumin 16 днів тому

    You are so good!!

  • @hzqhrmln
    @hzqhrmln 17 днів тому

    Very helpful! 😊

  • @mornmeetsthedew
    @mornmeetsthedew 17 днів тому

    Please make a video about DNA polymerase Domain in detail

  • @zackkykada567
    @zackkykada567 17 днів тому

    thank you so much for this explanation as i came from an article completely thinking its a hard thing to learn then met your video that gave me light on the process ps:name of the article is "day in a life of a spliceosome"

  • @ArjunRam
    @ArjunRam 18 днів тому

    Sir can you plz suggests the standrad book, where all the structure step wise mentioned 🙏

  • @tanishahalder9632
    @tanishahalder9632 20 днів тому

    Why didn't I come across this video earlier? You explain it so well and i immediately understood the concept. This channel is really underrated. Thank you so much.

  • @jahnvisingh5142
    @jahnvisingh5142 20 днів тому

    Great explanation, thank you so much from CSIR India lab👏🌷

  • @MrPuff1026
    @MrPuff1026 21 день тому

    the level of quality in your videos are amazing. thank you for your work.

  • @farhanalfiansyah5165
    @farhanalfiansyah5165 22 дні тому

    you help me out sir. thanks a lot.

  • @khurshaidanwar1580
    @khurshaidanwar1580 23 дні тому

    Sir, that is fantastic however 7:40 is gtgg

    • @theCrux
      @theCrux 22 дні тому

      Yep, good catch!

  • @sinannissar47
    @sinannissar47 28 днів тому

    Man u r crazy ur detailing, drawing explaining speed of video is 🫡

  • @pomme9728
    @pomme9728 Місяць тому

    WOW! You make it so clear, thank you!!

  • @lovekenmaforlife3406
    @lovekenmaforlife3406 Місяць тому

    thank you!!!

  • @aditikaushal1611
    @aditikaushal1611 Місяць тому

    The explanation was so good . Thanks for the video 🤍

  • @aflahshadilmp1818
    @aflahshadilmp1818 Місяць тому

    Amazing lecture sir. keep up the good work.🖤🖤

  • @chandandeosthali6625
    @chandandeosthali6625 Місяць тому

    Amazing board work and nice explanation!!! Thank you...

  • @arnabghosh676
    @arnabghosh676 Місяць тому

    Man..I love your videos...can you suggest any books for this topic??...It would be great if you suggest books in your descriptions.

  • @deepikaosmania
    @deepikaosmania Місяць тому

    Sir, I am a great fan of your videos . I have request. Can you please make a video on rRNA processing including ETS and ITS removal and other details

  • @sergiumitrea6437
    @sergiumitrea6437 Місяць тому

    Hello from România, what i can do if i remove 300 pear tree with crown gall? How much years i cant plant in that area? In romania the enginner say 5 years to no plant nothing in that areea . Thanks.

  • @lovekenmaforlife3406
    @lovekenmaforlife3406 Місяць тому

    yooo tysm!!!!!!

  • @jimmiosias3555
    @jimmiosias3555 Місяць тому

    thank you ❤

  • @AmnaYaseen-jq8rw
    @AmnaYaseen-jq8rw Місяць тому

    great well explained

  • @ZnZn-qo9ol
    @ZnZn-qo9ol Місяць тому

    Big thanks from China!!!! I can still get the other techniques by reading review articles, but struggled to understand this one on my own. Real life saver!!!

  • @genisessay
    @genisessay Місяць тому

    Bro i am serious, you are literally saving my life. Why stop, please keep going. The best explanation I've ever seen and its in great detail, its perfect...

  • @marcellasidella8419
    @marcellasidella8419 Місяць тому

    Sir, I am not clear how loop formation can solve the problem of incompatibility between primase and helicase. Could you please help me? 17:13

  • @kkmarskk
    @kkmarskk Місяць тому

    Crystal clear explanation, very helpful for exams

  • @mourifat8659
    @mourifat8659 Місяць тому

    Is there any lab in your knowledge which offers PhD position in core genetic engineering ?

  • @deonyz
    @deonyz Місяць тому

    Very educative description! Thank you!

  • @everythingisconnected1988
    @everythingisconnected1988 Місяць тому

    Amazingly clear video, thank you! Just a small remark for you: it's 'foreign', not 'foriegn' :)

  • @marygraceconcepcioncantale1640
    @marygraceconcepcioncantale1640 Місяць тому

    I love how you discuss these lessons, I am a bit of a visual learner and your videos help me a lot <3

  • @umaananth3602
    @umaananth3602 Місяць тому

    Excellent future effectors .........vpr's of 3 proteins called RTA - or synergistic activators

  • @AbidHasan007
    @AbidHasan007 Місяць тому

    Lots of thanks. Awesome! You made my day. Just a few minutes ago, I understood the whole topic and was ready for my exam .

  • @GromGoVrom
    @GromGoVrom Місяць тому

    I appreciate your ability to take all the information about this subject and package it into a thorough lecture. Thank you very much.

  • @antoineroquentin2852
    @antoineroquentin2852 Місяць тому

    Yes bro thank you so much! I’m also subbed on your patreon!

  • @theCrux
    @theCrux Місяць тому

    Epigenetic Replication: ua-cam.com/video/HB4IAiYqdzA/v-deo.html CRISPR-Cas9 in Genome Editing: ua-cam.com/video/7ESZTE6rjLI/v-deo.html CRISPR-KI CRISPR-KO: ua-cam.com/video/f0Q6JKu8Xjk/v-deo.html Base Editing: ua-cam.com/video/B1Bv-UmxugM/v-deo.html

  • @deepthijojikarivelil5054
    @deepthijojikarivelil5054 Місяць тому

    The Primase and helicase incompatibility was something that I struggled to understand! Thanks a lot for this video!!