The CRISPR/Cas system that evolved to protect bacteria and archaea from foreign DNA has rapidly been used by molecular biologists for a number of purposes. As discussed in Box 6-2, recombination in the cell initiates with a double-stranded break in the DNA sequence. Once that break has been made (and not repaired), the sequences at that site can be altered, deleted, or replaced by recombination. As noted in Tool Box 11-2, site-specific recombination is a widely used tool for editing genomes.
Recombination at double-stranded breaks allows a scientist to cleave DNA within a living cell by providing that strain with the appropriate nuclease – most commonly Cas9 from the well-studied CRISPR/Cas system of Streptococcus pyogenes – and a complementary RNA designed to look as though it has been transcribed and clipped off a CRISPR array.
The key to the editing is to target double-stranded break precisely. Think of how we edit a manuscript in a word processing program. We search for the part of the manuscript we want to edit, possibly by using the “Find” function to locate a particular word or string of characters. We then click on the mouse to define the exact positions of the edits we want to make. We then make the edits—corrections, insertions, deletions, and so on—at that site. This analogy works for understanding genome editing using CRISPR. Cas nucleases are proteins that cleave DNA after being guided to a target site by a complementary RNA. That is, the guide RNA is analogous to the Find function that locates the part of the genome to be edited. The Cas nuclease that makes a double-stranded break is the equivalent of the mouse click.
Cas9 and guide RNAs have been used in this way to edit genes in bacteria, invertebrates and a wide range of model animals, ranging from nematodes and flies to zebrafish and Cynomolgous monkeys. Figure A shows phenotypes affecting body shape, embryo structure and eye color that were made in worms, zebrafish and flies respectively using CRISPR.
Cas9 proteins have two catalytic domains, with each one cleaving one strand of the target DNA to produce a blunt-ended cleaved site. The catalytic domains of Cas9 require a lysine at position 10 and a histidine residue at position 840 for activity: a Cas9 derivative carrying D10A and H840A mutations is unable to cleave DNA to which it is targeted but can still bind it. This ‘dead Cas9’ (dCas9) prevents transcription by sterically hindering RNA polymerase and is employed for this CRISPR interference (CRISPRi) process. This method has been used in bacteria to block transcription of individuals since RNAi cannot be used.