The CRISPR/Cas9 technique is derived from a naturally occurring defense mechanism used by bacteria to defend themselves from viral infections and plasmids, by providing a type of acquired immunity for the bacteria. Over millenia as viruses have attempted to invade bacteria, the bacteria have responded by launching RNAs that match, attach to, and attack a recognized virus.
After the viral invasion is repelled, what remains in the bacterial DNA are short sequence repetitions (hence "palindrome") with short segments of spacer DNA in-between them. These repetitions and spacer DNA become built-up over time, after each episode versus bacterial viruses or plasmids.
In the early 2010's, researchers in different labs around the world finally understood this molecular defense mechanism. They then had a globally mutual Aha! moment when they realized this bacterial self-preservation tactic could be engineered to cut not only viral DNA, but any DNA sequence at a specifically selected gene or genes by altering guide RNAs in combination with an enzyme called Cas9 to match a targeted gene or genes.
Guide RNAs (sgRNA) are short synthetic oligonucleotide sequences that are complementary to a targeted DNA sequence. The sgRNA is part of a longer RNA molecule that forms a riboprotein with the Cas9 enzyme machinery. As the name implies, the guide RNA positions the Cas9 enzyme to the correct position on the target DNA for cleavage.
The end result is essentially a programmable nuclease that provides a gene editing technique for cutting double-stranded DNA at any desired location within a genome.
No less a visionary than Bill Gates recently said:
"In the area of biology, there's this new technology. It's called CRISPR, where you can actually go into a gene and either knock it out, or turn it on, or turn it off.
That's going to accelerate coming up with new medicines…understanding the human body. That's an amazing thing."
CRISPR/Cas9 provides significant improvements above and beyond previous protein-based gene editing methods such as Zinc-finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). CRISPR/Cas9 is less expensive and less time-consuming than ZFNs and TALENs, and is appreciably more reliable than ZFNs.
Even more importantly – unlike ZFNs and TALENs – CRISPR/Cas9 can be directed to multiple genes simultaneously, which is a major benefit when investigating complicated human diseases that originate not with a lone mutation, but by multiple mutating genes interacting with each other.
Thus... instead of dealing with one gene at a time, researchers can knock-out or turn on many genes at once. The "surgical" accuracy of CRISPR/Cas9 is a quantum leap beyond the previous techniques, and scientists can take the accuracy even farther by targeting exact nucleotides within those specific genes.
Simply put: it allows scientists to quickly determine gene editing efficiency in CRISPR/Cas9 efforts, as demonstrated in a series of experiments performed by Integrated DNA Technologies (IDT).
Measuring gene editing efficiency is essentially a function of measuring the transfection efficiency of the guided RNA and Cas9 protein. Transfection is a molecular mechanism for introducing foreign DNA into a cell, either by chemical or non-chemical methods.
Gene editing efficiency is also dependent on the performance of the chosen programmed repair mechanisms at the DNA cleavage site. Typically there are two types of repair mechanisms: non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ often results in the creation of small insertions and deletions that interrupt gene function. HDR uses homologous DNA as a means for DNA repair. With it, scientists can create many types of gene modifications determined by the introduction of donor DNA that has a desired sequence flanked on either side with homology to the target.
The point is this: whether reserchers are using NHEJ or HDR programmable repair mechanisms, the AATI Fragment Analyzer will rapidly determine the gene editing efficiency for CRISPR/Cas9 experiments.
Here's how. First, after a mutation insertion by the CRISPR/Cas9 system has occurred, DNA is extracted and amplified using PCR primers specific to the gene of interest. Next, heteroduplexed DNA is cleaved with an enzyme that recognizes the mismatched nucleotides of interest. Finally, Fragment Analyzer quickly and easily identifies those cleaved DNA fragments, which are indicative of the presence of new CRISPR/Cas9 gene mutations.
One of our customers – Integrated DNA Technologies – a major supplier of oligonucleotides to the biotech industry, and developer of gBlocks Gene Fragments for the CRISPR/Cas9 application – has conducted many CRISPR/Cas9 experiments on the Fragment Analyzer.
IDT found that:
"...analysis of gene editing events on the Fragment Analyzer allowed for an accurate, quantitative assessment of gene disruption in a rapid 96-well format and used one-tenth the mass of PCR product needed to visualize reaction on agarose gels."
"...This system simplifies gene targeting experiments and addresses the observed problem of low transfection efficiency. An optimized protocol was developed to transfect U6-sgRNA gBlocks Gene Fragments for CRISPR/Cas9-mediated editing of genomic DNA..."
"Use of the CRISPR/Cas9 method of gene editing can be streamlined by the availability of site selection algorithms to enrich for the most active single-guide RNAs (sgRNAs) from the many potential PAM/guide sites present in a gene of interest..."
"...CRISPR/Cas9 has recently emerged as a flexible, accurate, and costeffective system for genome modification that has rapidly increased in popularity over the past two years due to several advantages it holds over other methods. Recognizing the vast potential of this technique in creating disease models, researchers are busy developing new methods to build upon its strengths. Here we describe protocols that alleviate some of the practical issues still faced in using CRISPR/Cas9, particularly in high-throughput applications."
"...The CRISPR-associated RNA-guided nuclease Cas9 has emerged as a powerful tool for genome engineering in a variety of organisms. To achieve efficient gene targeting rates in Drosophila, current approaches require either injection of in vitro transcribed RNAs or injection into transgenic Cas9-expressing embryos. We report a simple and versatile alternative method for CRISPR-mediated genome editing in Drosophila using bicistronic Cas9/sgRNA expression vectors...."
"...Transcription activator-like effector nucleases (TALENs) emerged as powerful tools for locus-specific genomeengineering. Due to the ease of TALEN assembly, the key to streamlining TALEN-induced mutagenesis lies in identifying efficient TALEN pairs and optimizing TALEN mRNA injection concentrations to minimize the effort to screen for mutant offspring. Here we present a simple methodology to quantitatively assess bi-allelic TALEN cutting, as well as approaches that permit accurate measures of somatic and germline mutation rates in Drosophila melanogaster..."