Unformatted text preview: 1 Exam 3
Average score: 76
Highest score: 94.5 2 Which of the following is a marker for mesenchymal
E) Nkx2.1 3 What will happen after DNA double strand break in a
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
E) EMS mutagenesis
P element transpons
DNA homologues recombination
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
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
(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
- Spring '09
- DNA, CRISPR, McKenna, genome engineering, genome edi, Maddalo