History of CRISPR
The foundational discoveries leading to the development of CRISPR-Cas9 technology date back to 1987, when researchers first identified palindromic segments of DNA in bacteria. At the time, the significance of these Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), was not known.
In fact, it wasn’t until 2007 that researchers showed that CRISPR plays an important role in microbial innate immunity. Microbes, upon viral infection, deploy a special nuclease, directed by a guide RNA, to cleave specific sequences of the viral DNA. The excised DNA fragment may be stored to retain a genetic memory and disable future infections, similar to the array of antigens stored in our immune system for cellular defense against pathogens. In 2012, research groups of Doudna and Charpentier collaboratively deduced the cleavage mechanism of RNA-guided Cas9 and predicted its potential use in programmed genome editing.
In 2013, Feng Zhang’s group used the CRISPR system for genome editing in eukaryotic cells (human and mouse) for the first time. Thereafter, CRISPR has been harnessed as a common tool for editing genomes of various organisms in research projects across the globe.
CRISPR-based genome editing requires two components: a guide RNA and a CRISPR-associated endonuclease protein (Cas). The guide RNA, analogous to a GPS system, directs the Cas nuclease to the specific target DNA sequence, which then cuts the DNA at that site. The most commonly used nuclease, SpCas9, is the one isolated from the bacterium Streptococcus pyogenes.
The SpCas9 nuclease contains two protein lobes: a recognition lobe and a nuclease lobe. Upon binding the target DNA sequence with the help of the guide RNA, the recognition lobe interacts with the DNA strand to double check for complementarity. The nuclease lobe, similar to a pair of molecular scissors, then creates a double-strand break (DSB) in the target DNA.
Once a DSB is created in the DNA, the cell tries to repair it via non-homologous end to end joining (NHEJ), which is a quick-fix repair mechanism for ligation of the blunt ends of DNA and is prone to errors. The process often results in insertions or deletion of bases (indels), which can lead to protein disruption, and is the preferred pathway for knocking out a particular gene.
Note that while NHEJ yields knockouts, CRISPR can also be used to knock-ins desired sequences at specific loci in the cellular genome via the homology-directed pathway, HDR.
Mechanism of CRISPR-mediated Knockouts: The guide RNA (sgRNA in the schematic stands for single guide RNA) directs the Cas9 nuclease to its target DNA. Cas9 creates a double-strand break in the DNA, which the cell repairs using one of its two natural mechanisms. NHEJ is a quick repair that involves a simple end-to-end joining of the nicked ends, often resulting in insertions or deletions that may generate knockouts.
CRISPR Experimental Workflow
One of the most critical steps of a CRISPR experiment is designing efficient and specific guide RNAs, which is facilitated by the availability of state-of-the-art design tools.
Choosing the delivery format of the CRISPR components is the next step in the CRISPR workflow. Transfecting plasmids bearing the guide RNA sequence and nuclease encoding the Cas nuclease sequence has been conventionally used in genome engineering. In vitro transcribed RNAs (IVT) have also been used widely in CRISPR experiments.
However, both these delivery systems suffer from low efficiency and labor-intensive methods. The availability of economical, high-quality synthetic guide RNA allows delivery of CRISPR components complexed in a ribonucleoprotein (RNP) format, which enables highest editing efficiencies and the most reproducible CRISPR results. The RNP format is now the preferred choice of many researchers for genome engineering experiments.
Once the guide RNA is transfected into cells, the editing efficiency is analyzed using tools such as ICE.
Schematic of the CRISPR workflow. The process for CRISPR mediated genome editing includes the crucial step of designing guide RNAs, introducing them into cells, and finally analyzing the editing efficiency.
Guided Edit Interactive Tool
Meet Guided Edit, an interactive tool to help you navigate through a clear-cut decision tree, so you arrive at the best solution for your CRISPR editing goal. Start by telling us a bit about your project and answer a few questions.