Genome editing. Knock-ins
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1 Genome editing Knock-ins
2 Experiment design?
3 Should we even do it? In mouse or rat, the HR-mediated knock-in of homologous fragments derived from a donor vector functions well. However, HR-dependent knock-in events are restricted in zebrafish. Explanation: initial mitoses of zebrafish embryonic cleavages occur faster than those of mouse???
4 1) Choose a technique zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system
5 2) Choose a repair mechanism:
6 Single-stranded oligonucleotide template insertion: Introduction of small genomic alternations: Single nucleotide alternation (mutation) Introduction of restriction site Introduction of LoxP site Introduction of small protein tags (e.g. HA) Homologous sequence requirement: bp Longer arm less recombination events(?) Undesired mutations: indel mutations; tandem integration of ssodns
7 Single-stranded oligonucleotide template insertion:
8 3) Specifics A) What guide?
9 B) How long should the arm be?
10 Integration of Donor DNA by Homologous Recombination Insertion of a bigger DNA fragments though addition of a longer homology arms (~1000bp).
11 Again: B) How long should the arm Length of the arms: be? The number of the donor molecules can influence the efficiency of HR when the amount of the donors injected into the embryos is low. However, if excess amount of donor molecules is injected, the efficiency of HR will no longer increase. 1.5% germline transmission frequency all showed DNA integration in a precise and targeted manner
12 C: Circular or Linear? kjk How translatable is TALEN experiments for CRISPR??? 10% germline transmission frequency all showed DNA integration in a precise and targeted manner
13 More questions???
14 D: If circular how long should the arm be? Higher percentage of sfgfp-positive embryos in the L group, compared with the M and S groups
15 E: Should the vector be cut inside? Internal cut in shorter homology arm can increase the frequency of recombination? not only the length of homology arm, but also the configuration of the targeting construct can influence the frequency of HR events in somatic tissue.
16 E: Should the vector be cut inside? Effect on transission
17 Insertion of Donor DNA by Non- Homologous End Joining (NHEJ) The targeted genomic locus and the donor vector containing the TALEN/CRISPR target sequences were simultaneously cleaved by TALEN or Cas9. The simple construction of the donor vector because there is no requirement for long homology arms. knock-in donors as large as 5.7 kb
18 Insertion of Donor DNA by Non- Homologous End Joining (NHEJ) Mutations: 50% exhibited small deletions (24/48 sequences) and 33% small insertions (16/48), while 17% (8/48) corresponded to ligation of nonmodified DNA sequences from the targeted locus and plasmid (perfect repair). Injection of linearized plasmid is more toxic and less efficient.
19 F : Homology arms? Other problems? Mutations: 50% exhibited small deletions and 33% small insertions, while 17% corresponded to ligation of non-modified DNA sequences (perfect repair). Injection of linearized plasmid is more toxic and less efficient. The major homology-independent mechanism of DSB repair, is at least 10-fold more active than HR during early zebrafish development (the germline transmission rate of forward donor DNA integration was ~25%).
20 Integration of Donor DNA by Microhomology-Mediated End Joining (MMEJ) Insert is targeted to the appropriate site by microhomology sequences (5-25 bases). Increase the efficiency of insertion in comparison to NHEJ (85 vs. 53%). Increased number of precise targeted integration (60% 77%) vs. ~20%.
21 G: Again homology arm?
22 H: Screen at every step?
23 A single RNA-guided endonuclease Cpf1 Feature Cas9 Cpf1 Structure 2 RNA 1 RNA Cutting mechanism Blunt end cut Staggered end cuts Cutting site Proximal to recognition site Distal from recognition site Target site G-rich PAM T-rich PAM
24 Should we even do it? In mouse or rat, the HR-mediated knock-in of homologous fragments derived from a donor vector functions well. However, HR-dependent knock-in events are restricted in zebrafish. Explanation: initial mitoses of zebrafish embryonic cleavages occur faster than those of mouse???
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