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--- chapter_17 [2025/05/16 21:50] – [Conditional knockouts] mike
+++ chapter_17 [2025/05/16 21:52] (current) – [Viral vectors] mike
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 ===== CRISPR/Cas9 =====
-As we learned in [[chapter_16|Chap. 16]], homologous recombination occurs only at low frequencies in mammalian cells – this is why it was necessary to develop technologies to culture ES cells in vitro, so that we could examine large numbers of ES cells to look for rare events. It turns out that the frequency of homologous recombination increases enormously if there is a double-stranded break in the DNA near the site you wish to have homologous recombination. CRISPR/Cas9 (Fig. {{ref>Fig4}}) is an enzyme usually isolated from the bacterium //Streptococcus pyogenes// that can be used to cleave dsDNA to generate a double-stranded break. However, unlike restriction enzymes that only cut short defined palindromic sequences ([[chapter_09|Chap. 9]]), it is possible to modify CRISPR/Cas9 such that we can essentially make it cut almost any unique DNA sequence we wish. This is possible because CRISPR/Cas9 uses a guide RNA (gRNA) as part of its enzyme structure and the gRNA sequence is what guides it to its target DNA though RNA/DNA base pairing. It is relatively easy to modify this RNA sequence so that CRISPR/Cas9 will target the DNA sequence of a specific gene you are interested in. When dsDNA is cleaved in live cells, repair mechanisms will rejoin the cleaved DNA; however, errors often occur during this process, and indel mutations usually occur and therefore frameshifts, which usually creates null alleles. If a targeting construct is also present, homologous recombination will occur at a very high frequency; this allows for creation of knock-in alleles without necessarily using the Cre-$lox$-based strategy described above.
+As we learned in [[chapter_16|Chap. 16]], homologous recombination occurs only at low frequencies in mammalian cells – this is why it was necessary to develop technologies to culture ES cells in vitro, so that we could examine large numbers of ES cells to look for rare events. It turns out that the frequency of homologous recombination increases enormously if there is a double-stranded break in the DNA near the site you wish to have homologous recombination. CRISPR/Cas9 (Fig. {{ref>Fig4}}) is an enzyme usually isolated from the bacterium //Streptococcus pyogenes// that can be used to cleave dsDNA to generate a double-stranded break. However, unlike restriction enzymes that only cut short defined palindromic sequences ([[chapter_09|Chap. 9]]), it is possible to modify CRISPR/Cas9 such that we can essentially make it cut almost any unique DNA sequence we wish. This is possible because CRISPR/Cas9 uses a guide RNA (gRNA) as part of its enzyme structure and the gRNA sequence is what guides it to its target DNA through RNA/DNA base pairing. It is relatively easy to modify this RNA sequence so that CRISPR/Cas9 will target the DNA sequence of a specific gene you are interested in. When dsDNA is cleaved in live cells, repair mechanisms will rejoin the cleaved DNA; however, errors often occur during this process, and indel mutations usually occur and therefore frameshifts, which usually creates null alleles. If a targeting construct is also present, homologous recombination will occur at a very high frequency; this allows for creation of knock-in alleles without necessarily using the Cre-$lox$-based strategy described above.
 <figure Fig4>
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   * A third plasmid that expresses the essential E2A, E4, and VA genes from adenovirus (green in Fig. {{ref>Fig6}})
-You would then introduce all three plasmids into cultured cells (such as HEK293 cells, a human embryonic kidney cell line). The purple and green plasmids, together with the HEK293 host cell genome, provide all the proteins needed to replicate and package newly replicated viral ssDNAs. However, only DNA with ITRs will be packaged. This means that the plasmids containing AAV and adenoviruses will not be packaged (they won't even be replicated) - only the ssDNA with the ITRs, and therefore your trangene, will be packaged into new viral capsids. You can harvest the rAAVs from the media of the HEK293 cell culture.
+You would then introduce all three plasmids into cultured cells (such as HEK293 cells, a human embryonic kidney cell line). The purple and green plasmids, together with the HEK293 host cell genome, provide all the proteins needed to replicate and package newly replicated viral ssDNAs. However, only DNA with ITRs will be packaged. This means that the plasmids containing AAV and adenoviruses will not be packaged (they won't even be replicated) - only the ssDNA with the ITRs, and therefore your transgene, will be packaged into new viral capsids. You can harvest the rAAVs from the media of the HEK293 cell culture.
 <figure Fig6>
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 </columns>
 <caption>
-Some example of AAV serotypes (variants) with different tissue targeting specificity (tropism). Adapted from Naso et al. (2017) BioDrugs 31(4): 317-334 https://doi.org/10.1007%2Fs40259-017-0234-5. Liscensing: [[http://creativecommons.org/licenses/by-nc/4.0/|CC BY-NC-4.0]].
+Some example of AAV serotypes (variants) with different tissue targeting specificity (tropism). Adapted from Naso et al. (2017) BioDrugs 31(4): 317-334 https://doi.org/10.1007%2Fs40259-017-0234-5. Licensing: [[http://creativecommons.org/licenses/by-nc/4.0/|CC BY-NC-4.0]].
 </caption>
 </table>