Early studies in Caenorhabditis elegans and Drosophila melanogaster saw large-scale, systematic loss of function (LOF) screens performed through saturation mutagenesis, demonstrating the potential of this approach to characterise genetic pathways and identify genes with unique and essential functions. The saturation mutagenesis technique was later applied in other organisms, for example zebrafish and mice.
Targeted approaches for gene knockdown emerged in the 1980s with techniques such as homologous recombination, trans-cleaving ribozymes, and antisense technologies.
By the year 2000, RNA interference (RNAi) technology had emerged as a fast, simple, and inexpensive technique for targeted gene knockdown, and was routinely being used to study in vivo gene function in C. elegans. Indeed, in the span of only a few years following its discovery by Fire et al. (1998), almost all of the ~19,000 genes in C. elegans had been analysed using RNAi-based knockdown.
The production of RNAi libraries facilitated the application of this technology on a genome-wide scale, and RNAi-based methods became the predominant approach for genome-wide knockdown screens.
Nevertheless, RNAi-based approaches to genome-wide knockdown screens have their limitations. For one, the high off-target effects cause issues with false-positive observations. Additionally, because RNAi reduces gene expression at the post-transcriptional level by targeting RNA, RNAi-based screens only result in partial and short-term suppression of genes. Whilst partial knockdown may be desirable in certain situations, a technology with improved targeting efficiency and fewer off-target effects was needed.
Since initial identification as a prokaryotic adaptive immune system, the bacterial type II clustered regularly interspaced short palindrome repeats (CRISPR)/Cas9 system has become a simple and efficient tool for generating targeted LOF mutations. It has been successfully applied to edit human genomes, and has started to displace RNAi as the dominant tool in mammalian studies. In the context of genome-wide knockout screens, recent studies have demonstrated that CRISPR/Cas9 screens are able to achieve highly efficient and complete protein depletion, and overcome the off-target issues seen with RNAi screens. In summary, the recent emergence of CRISPR-Cas9 has dramatically increased our ability to perform large-scale LOF screens. The versatility and programmability of Cas9, coupled with the low noise, high knockout efficiency and minimal off-target effects, have made CRISPR the platform of choice for many researchers engaging in gene targeting and editing.