CRISPR, an acronym for clustered regularly interspaced short palindromic repeats, is a family of genes that first evolved in prokaryotic organisms such as bacteria and archaea to defend against infectious phages.1 Analogous to eukaryotic adaptive immune memory, CRISPR sequences derive from bacteriophages that previously infected prokaryotes; bacteria use their CRISPR sequences and nucleases called CRISPR associated (Cas) proteins to detect and destroy familiar bacteriophages.1 Today, researchers build on the mechanisms of prokaryotic CRISPR systems to engineer CRISPR-Cas mediated gene editing technologies, which use Cas proteins and guide RNAs to block, cut, or edit target genes.1
How Gene Editing Works
Originating from the bacterium Streptococcus pyogenes, Cas9 was the first Cas protein that scientists repurposed for gene editing.1 CRISPR-Cas9 technology uses a single guide RNA (sgRNA) to target and cleave DNA. Researchers engineer target-specific sgRNAs by combining two RNA molecules from the bacterial CRISPR system: a sequence that recognizes a specific location in the DNA (crRNA) and a sequence that acts as a binding scaffold for Cas9 (tracrRNA).1 The modifiable sgRNA sequence allows scientists to program a CRISPR-Cas9 system to target any DNA sequence of interest if it is near a Cas-specific DNA sequence called a protospacer-adjacent motif (PAM).1,2 The PAM initiates Cas9-DNA binding, the sgRNA invades the double helix and hybridizes with the target DNA, and Cas9 breaks the unwound double-stranded target DNA immediately in front of the PAM. Repair mechanisms, namely homology-directed repair (HDR) and nonhomologous end joining (NHEJ), repair the break, which can alter the target gene’s biological function.3
For instance, researchers can use the different repair mechanisms to their advantage to intentionally insert a desired sequence change via template-dependent HDR or introduce random changes through template-independent NHEJ. If scientists provide the cellular repair machinery with a target gene template that contains a mutation, such as a disease-relevant mutation, the HDR process will incorporate the templated mutation into the genome after Cas9 cleavage.1,2 In contrast, the more error prone NHEJ pathway repairs the Cas9 cut without a template by introducing random insertions and deletions (indels) that ultimately disrupt gene expression or lead to loss of function.1,2 Either of these approaches enable researchers to investigate how silencing or editing a targeted gene affects its downstream pathways.1
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