Scientists have had the knowledge and ability to edit genomes for many years, but CRISPR technology has brought major improvements to the speed, cost, accuracy, and efficiency of genome editing. The history of genome editing technologies shows the remarkable progress in this field and also relays the critical role that basic science research plays in the development of research tools and potential disease treatments.Homologous recombinationThe earliest method scientists used to edit genomes in living cells was homologous recombination. Homologous recombination is the exchange (recombination) of genetic information between two similar (homologous) strands of DNA.1 Scientists began developing this technique in the late 1970s following observations that yeast, like other organisms, can carry out homologous recombination naturally.To perform homologous recombination in the laboratory, one must generate and isolate DNA fragments bearing genome sequences similar to the portion of the genome that is to be edited. These isolated fragments can be injected into individual cells or taken up by cells using special chemicals. Once inside a cell, these DNA fragments can then recombine with the cell's DNA to replace the targeted portion of the genome.This type of homologous recombination is limited by the fact that it is extremely inefficient in most cell types. This technique can have as low as a one-in-a-million probability of successful editing. Another weakness of homologous recombination is that it is inaccurate and has a high rate of error when the injected DNA fragments insert into an unintended part of the genome, causing what are known as off-target edits.2Zinc-finger nucleases (ZFN)In the 1990s researchers started using zinc-finger nucleases (ZFN) to improve the specificity of genome editing and reduce off-target edits. The structures of ZFNs are engineered from naturally-occurring proteins that were discovered in eukaryotic organisms. Scientists can engineer these proteins to bind to specific DNA sequences in the genome and cut DNA. Once bound to their target DNA sequence, the ZFNs cut the genome at the specified location, allowing scientists to either delete the target DNA sequence or replace it with a new DNA sequence via homologous recombination.Although ZFNs improved the success rate of genome editing to about 10 percent, it is difficult and time-consuming to design, construct, and produce successful zinc finger proteins, and a new ZFN must be engineered for each new target DNA sequence.Transcription activator-like effector nucleases (TALENs)In 2009, a new class of proteins called Transcription Activator-Like Effector Nucleases (TALENs) arrived to the genome editing scene. Similar to ZFNs, transcription activator-like effector nucleases (TALENs) are engineered from proteins found in nature and are capable of binding to specific DNA sequences.While TALENs and ZFNs are comparable in terms of how efficiently they can create edits to the genome, TALENs bear the advantage of greater simplicity. It is much easier to engineer TALENs than it is to synthesize ZFNs.3Clustered regularly interspaced short palindromic repeats (CRISPR)Though ZFN and TALEN technology increase the specificity and efficiency of genome editing, they are relatively expensive and complicated to use in the lab. Each edit would require the construction of a new ZFN or TALEN protein, and engineering proteins can be a difficult process that is prone to error. This is one reason why CRISPR is a game-changing technology; unlike its predecessors, CRISPR is a simple technology with little assembly required. CRISPR associated DNA sequences were first observed in bacteria in the early 1990s, but it was not until the 2000s that the scientific community understood its ability to recognize specific genome sequences and cut them via the Cas9 protein, a protein that works with CRISPR and that has DNA-cutting abilities. In nature, CRISPR is used by bacteria as an immune system to kill invading viruses, but it has now been adapted for use in the lab. 4With CRISPR, researchers create a short RNA template that matches a target DNA sequence in the genome. Creating synthetic RNA sequences is much easier than engineering proteins as is those required for ZFNs and TALENs. Strands of RNA and DNA can bind to each other when they have matching sequences. The RNA portion of the CRISPR, called a guide RNA, directs Cas9 enzyme to the targeted DNA sequence. Cas9 cuts the genome at this location to make the edit. CRISPR can make deletions in the genome and/or be engineered to insert new DNA sequences. One group of scientists found that CRISPR is six times more efficient than ZFNs or TALENs in creating targeted mutations to the genome.5 Large-scale genomics projects that once took many years and tens of thousands of dollars can now be completed at a small fraction of time and price.