Curing Rare Diseases — 2

CRISPR technology is rather famous these days, but what is it exactly, and where did it come from? In the 1980s, sequencing the genes of bacteria led to the discovery of unusual DNA stretches that were called CRISPR and were later found to be part of bacteria’s defense system against viruses. The unusual palindromic repeat stretches of bacteria DNA were coded for RNA that matched invading virus genes and helped destroy them. This natural defense system has been around for billions of years. But in 2012, a paper in Science with the arcane title “A Programmable Dual RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity” woke us up to a new possibility in life science: CRISPR could precisely edit DNA in test-tube experiments using a guide RNA and the Cas9 molecular scissors enzyme. (Cas stands for CRISPR-associated proteins.) Like the seminal 2005 discovery by Katalin Karikó and Drew Weissman for blocking mRNA-induced in vivo inflammation, it attracted little notice initially, but later it was the basis for a Nobel Prize awarded to Jennifer Doudna and Emmanuelle Charpentier. Within a year of the CRISPR discovery, there were multiple reports of precise DNA editing in animal and human cells.

Before CRISPR, we had tools that cleaved DNA nucleotides known as nucleases to do genome editing, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), but they were difficult to program to achieve precise editing.

Over the next decade, CRISPR technology underwent intensive refinement to improve the accuracy and precision of editing, reducing the chance for off-target effects. All three of these nucleases (ZFNs, TALENs, CRISPR-Cas 9) cut the double-stranded DNA into two pieces. The cell wants to repair the broken ends of chromosomes, which it can do accurately about 90 percent of the time. That leaves a small percentage of mistakes in the rejoining repair process. But the nuclease keeps cutting, and the repair process keeps following, accumulating more mistakes. The cycle continues until the nuclease no longer recognizes the altered, mistake-laden sequence, with nucleotide insertions and deletions, ultimately achieving disruption of the gene. The downside from this double-strand break and repair process is the lack of control of the resultant sequence with random insertion and deletions (fig. 8.1). It’s a disruptive gene-knockout strategy that can’t be used to fix a gene. Let’s call this CRISPR 1.0.