The technique for obtaining knockdowns with CRISPR-Cas9 entails the use of proteins known as CRISPR-associated genes, which can be programmed to insert exogenous DNA fragments into a CRISPR repeat locus. This method allows for the production of small RNAs that can serve as a template for Cas proteins to silence that same sequence in a cell. CRISPR is commonly used by many researchers for obtaining knockdowns in various mouse models, for the purpose of fine-tuning their genetic research. The main goal is usually to find the precise mechanism through which the silenced genes work, and what happens when their expression is diminished or stopped.
It is probably fair to say that CRISPR-Cas9 has already leapfrogged the previous technologies such as TALEN、CRISPRi (CRISPR interference) and RNAi, mostly because CRISPR-Cas9 offers an unprecedented simplicity to target a large variety of functional domains to various genomic sites. Perhaps the most striking difference among these various approaches is that RNAi and CRISPRi induce transient knockdown of gene expression, whereas genome editing via Cas9 endonuclease can induce permanent damage of the target DNA, leading to a null phenotype in some cases. Therefore, a major question is "do you want to generate a complete knockout or a hypomorphic knockdown?"
Aside from CRISPR, knockdown genes can also be obtained through various other means. TALENs and RNA interference are two of the best known methods, and they also enjoy nearly as much popularity as the CRISPR technique. Knockdown genes can be obtained with the help of oligonucleotide that temporarily bind to a gene, leading to a temporary or "transient" knockdown. Transient knockdown methods are commonly used in developmental biology, and are frequently referred to as "reverse genetics" techniques. They involve a number of possible options, such as gene binding, the blocking of mRNA translation or the use of siRNA (small interfering RNA).
RNA interference uses mRNA degradation to obtain silencing effects in specific genes. This is primarily a research tool that allows for easy screening, and makes it possible for researchers to determine what area of genetic research to focus on next. TALENs is another common method of obtaining knockdowns, which however, focuses on genome manipulation with the help of transcription activator-like effector nucleases (TALENs). With the help of CRISPR, knockdown genes are easier to obtain than ever before, and the technique shows great promise for future study.
Knockdown genes can be obtained either through genetic manipulation, through CRISPR, or with reagents such as RNA oligonucleotide or a short DNA. In comparison to other acute knock-down methods, CRISPR-Cas9-mediated knockdown is either more efficient and complete (e.g. RNA-interference based approaches) or faster (generation of conditional knockout mice and subsequent transfection with Cre), making it the ideal candidate for the investigation of neuronal function. While not always capable of disrupting gene function completely, hypomorphic knockdowns can offer a number of advantages over knockouts. Gene knockdown via RNAi or CRISPRi does not depend on frame shift mutations or ploidy.
Furthermore, partial gene knockdown allows researchers to study the function of essential genes whose knockout would otherwise be cell-lethal. Additionally, applications such as drug target discovery could benefit from phenotypic hypomorphs, since an RNAi or CRISPRi knockdown-rather than a knockout-may mimic the effects of an inhibitive drug more closely. However, in some cases, a full knockout may represent an idealized loss-of-function phenotype for the most potent inhibitive drugs. Moreover, gene knockdowns are reversible, a feature that has proven very useful in the past.
1. Boettcher and Mc Manus. Choosing the Right Tool for the Job: RNAi, TALEN or CRISPR. Mol Cell. 2015 May 21; 58(4): 575–585.
2. Tang et al. CRISPR/Cas9-mediated genome editing induces gene knockdown by altering the pre-mRNA splicing in mice. BMC Biotechnology. 2018;18:61.