Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 157

Bacteria and archaea possess an endogenous system of defense in which short single-stranded guide RNAs (sgRNAs) are used to target and destroy invading DNA (Volume II, Chapter 3, Box 3.9). One embodiment of this defense, the CRISPR-Cas9 (clustered regularly interspersed short palindromic repeat [CRISPR]-associated nuclease 9) system, has been adapted for effective and efficient targeting gene disruptions and mutations in any genome. The specificity depends on the ability of the sgRNAs to hybridize to the correct DNA sequence within the chromosome. Once annealed, the endonuclease Cas9 catalyzes formation of a double-strand break, which is then repaired, creating frameshifting insertion/deletion mutations within the gene. One advantage of using CRISPR-Cas9 methodology to modify cell genomes is that the method can be applied to any cell type. Like siRNAs, CRISPR-Cas9 can be used to affect individual mRNAs or to carry out genome-wide screens to identify cell genes that stimulate or block viral reproduction (Fig. 3.13). As with RNAi screens, the most convincing confirmation of the result is restoration of the phenotype by expression of a gene containing a mutation that makes it resistant to Cas9, via changes in the sgRNA target sequence.

Figure 3.13 Use of RNAi, haploid cells, and CRISPR-Cas9 to study virus-host interactions. In arrayed screens, siRNAs are introduced into cells growing in wells that are subsequently infected with virus. Production of infectious virus or a viral protein is quantified by plaque assay or measurement of a fluorescent protein. Individual siRNA with the desired effect can be identified based on their location in the multiwell plate. In pooled RNAi screens, collections of shRNA producing lentiviral vectors are used to infect cells. After selection for cells with integrated vectors, the cells are infected with the test virus and the production of a viral protein or infectious virus is monitored. In pooled haploid cell screens, cells are infected with lentiviruses at a low multiplicity of infection so that on average one viral genome integration per cell takes place. In pooled CRISPR-Cas9 screens, libraries of sgRNAs are introduced, via lentivirus vector, into cells that produce Cas9. After selection for lentiviral integration, cells are infected with virus. Cell survival and production of infectious virus or a viral protein may be measured depending on what types of genes are sought (e.g., those that are essential for reproduction). In each screen, the cell gene that is disrupted is identified by nucleotide sequencing.

While the experimental use of RNAi can lead to reduced protein production, genomic manipulation by CRISPR-Cas9 has advantages of complete depletion of the protein through the production of a homozygous null genotype and fewer off-target effects. With CRISPR-Cas9, the expression of a gene can be permanently extinguished. In contrast, the shRNA-expressing provirus must continually silence the product of ongoing transcription.