Lecture+17_2017 - 1 Exam 3 Average score 76 Highest score...

Info icon This preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: 1 Exam 3 Average score: 76 Highest score: 94.5 2 Which of the following is a marker for mesenchymal cells? A) E-Cad B) N-Cad C) Sox17 D) Vimentin E) Nkx2.1 3 What will happen after DNA double strand break in a cell? A) It will be repaired B) The cell will die C) Nothing happens 4 1.  Why genome engineering? 2.  What is CRISPR-Cas9? 3.  How to use CRISPR-Cas9 for genome [email protected]? 4.  [email protected] - cancer modeling - lineage tracing 5 Goal: make precise, targeted changes to the genome of living cells Which of the following is a targeted way to change a genome? A) B) C) D) E) EMS mutagenesis P element transpons DNA homologues recombination RNAi C and D 6 error-prone: inserCons, dels NHEJ specific sequence guided HR 7 zinc finger nuclease TALEN 8 9 1.  Why genome engineering? 2.  What is CRISPR-Cas9? 3.  How to use CRISPR-Cas9 for genome [email protected]? 4.  [email protected] 10 An adapCve immune system in bacteria: CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats Cas: CRISPR associated repeat spacer (incorporated invading DNA) 11 12 PAM sequence: 5’-NGG-3’ 13 1.  Why genome engineering? 2.  What is CRISPR-Cas9? 3.  How to use CRISPR-Cas9 for genome [email protected]? 4.  [email protected] 14 Jennifer Doudna Emmanuelle CharpenCer Feng Zhang UC Berkeley Germany MIT 15 Single guide RNA (sgRNA) combines the crRNA and tracrRNA into one RNA Steps to do CRISPR-Cas9 genome ediCng: with a linker loop. It forms a complex with Cas9 protein and recruits it to specific genomic DNA sequence. The targeting specificity comes from the base-paring between the crRNA sequence and target genomic sequence. 1. Look for N20-NGG sequence in your gene of interest 2. Design your single-guide RNA (target sequence + tracrRNA scaffold) 3. Deliver single-guide RNA and Cas9 into cells 16 17 a. Design the sgRNA sequence so that a potential double strand break point is near that point mutation in the genome. b. Generate sgRNA plasmid and obtain Cas9 plasmid. c. Use in vitro transcription for these plasmids to obtain sgRNA and Cas9 mRNA. d. Synthesize a DNA oligo template that contains the point mutation sequence flanked by the normal KRas gene sequence. e. Inject sgRNA, Cas9 mRNA, and the DNA template into mouse zygotes. f. Transfer to pseudopregnant female. g. After birth, do genotyping to confirm the desired genomic change has occurred. (Note: the traditional way to achieve this is to make a knock-in mouse using ES cells and chimeras, which is more laborious.) 18 How to use CRISPR to make KO/KI animals 1.  Why genome engineering? 2.  What is CRISPR-Cas9? 3.  How to use CRISPR-Cas9 for genome [email protected]? 4.  [email protected] - cancer modeling - lineage tracing 19 Eml4-Alk gene fusion is present in a subset of non-small cell lung cancers Maddalo et al, 2014 Nature 20 Maddalo et al, 2014 Nature 21 Delivery method: Package plasmids expressing Cas9 and two sgRNA into adenoviruses InsCll viruses into mouse lungs negaCve control Maddalo et al, 2014 Nature 22 Overall idea: Each cell has an engineered unique barcode that is editable by CRISPR/Cas9. Sequencing the barcodes to reconstruct history of cell lineage. McKenna, et al, 2016 Science 23 Procedures: Pafern of ediCng in an 30 hpf embryo McKenna, et al, 2016 Science 24 McKenna, et al, 2016 Science 25 Gene knockout/knockin in zygotes, cells, Cssues of virtually any animals Introduce specific point mutaCon/chromosome rearrangement in disease modeling Barcode for lineage tracing Use sgRNA libraries for high-throughput cell/Cssue screening Microscopic visualizaCon of specific genomic loci Enhance specific gene expression Correct congenital mutaCons in human embryos ? 26 Findings in the paper: Specific gene (β-globin) can be targeted in human zygotes Homologous recombinaCon directed repair is not efficient Edited embryos were mosaic Off-target effect is a problem 27 ...
View Full Document

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern