Figure 1: The current genome engineering technologies allow scientists to introduce double stranded breaks at specific sequences. Learn more on Addgene's Genome Engineering page.
The last few months have been an exciting time for genome engineering technology. A new system offers the first alternative to the current protein-based targeting (TALEN and Zinc Finger) methods used to specify a gene (or other DNA sequence). This new system uses a short RNA to guide a nuclease to the DNA target. This is CRISPR technology (Figure 1).
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) system was first discovered in bacteria and functions as a defense against foreign DNA, either viral or plasmid. So far three distinct bacterial CRISPR systems have been identified, termed type I, II and III. The Type II system is the basis for the current genome engineering technology available and is often simply referred to as CRISPR.
In bacteria, the endogenous CRISPR/Cas system targets foreign DNA with a short, complementary single-stranded RNA (CRISPR RNA or crRNA) that localizes the Cas9 nuclease to the target DNA sequence. The DNA target sequence can be on a plasmid or integrated into the bacterial genome. The DNA target also does not need to be unique and can appear in multiple locations, all of which will be targeted by the Cas9 nuclease for cleavage. The crRNA can bind on either strand of DNA and the Cas9 will cleave both strands (double strand break, DSB). The DSB results in the silencing of that DNA sequence.
The crRNA targeting sequences are transcribed from DNA sequences known as protospacers. Protospacers are clustered in the bacterial genome in a group called a CRISPR array. The protospacers are short sequences (~20bp) of known foreign DNA separated by a short palindromic repeat and kept like a record against future encounters. To create the CRISPR targeting RNA (crRNA), the array is transcribed and the RNA is processed to separate the individual recognition sequences between the repeats. In the Type II system, the processing of the CRISPR array transcript (pre-crRNA) into individual crRNAs is dependent on the presence of a trans-activating crRNA (tracrRNA) that has sequence complementary to the palindromic repeat. When the tracrRNA hybridizes to the short palindromic repeat, it triggers processing by the bacterial double-stranded RNA-specific ribonuclease, RNase III. Any crRNA and the tracrRNA can then both bind to the Cas9 nuclease, which then becomes activated and specific to the DNA sequence complimentary to the crRNA (Figure 2).
There are some restrictions to this new genome engineering system. Any potential target sequence must have a specific sequence on its 3’ end (the protospacer adjacent motif, PAM). The PAM is present in the DNA to be degraded but not the crRNA that’s produced to target it. The Type II CRISPR system is currently limited to target sequences that are N12-20NGG. Where NGG represents the PAM. Additionally, it is hypothesized that certain target sequences are believed to be problematic due to the RNA secondary structure they form. Interactions between the Cas9, tracrRNA and crRNA secondary structures are still poorly understood.
Figure 2: An overview of the endogenous Type II bacterial CRISPR/Cas system. Within the bacterial genome, a CRISPR array contains many unique protospacer sequences that have homology to various foreign DNA (e.g. viral genome). Protospacers are separated by a short palindromic repeat sequence. (A) The CRISPR array is transcribed to make the pre-CRISPR RNA (pre-crRNA). (B) The pre-crRNA is processed into individual crRNAs by a special trans-activating crRNA (tracrRNA) with homology to the short palindromic repeat. The tracrRNA helps recruit the RNAse III and Cas9 enzymes, which together separate the individual crRNAs. (C) The tracrRNA and Cas9 nuclease form a complex with each individual, unique crRNA. (D) Each crRNA:tracrRNA:Cas9 complex seeks out the DNA sequence complimentary to the crRNA. In the Type II CRISPR system a potential target sequence is only valid if it contains a special Protospacer Adjacent Motif (PAM) directly after where the crRNA would bind. (E) After the complex binds, the Cas9 separates the double stranded DNA target and cleaves both strands after the PAM. (F) The crRNA:tracrRNA:Cas9 complex unbinds after the double strand break.
The CRISPR systems that several research groups have developed for use in eukaryotic cells use a variation of a bacterial Cas9 nuclease that has been codon-optimized for their desired cell type. Additionally, a number of research groups have successfully demonstrated the effectiveness of a single fused crRNA-tracrRNA construct that functions with their codon-optimized Cas9 (Figure 3). This single RNA is often referred to as a guide RNA or gRNA. Within a gRNA, the crRNA portion is identified as the ‘target sequence’ and the tracrRNA is often referred to as the ‘scaffold’. This system has been shown to function in a variety of eukaryotic cells, including human, zebra fish and yeast. And for all of you interested in editing bacterial genomes, customized bacterial CRISPR systems have been created by the Doudna lab and the Marraffini lab to allow you to alter your favorite bacterial gene or DNA element.
There are many available online resources for helping scientists determine suitable target sites in their desired DNA sequence. The Church lab has published a bioinformatically generated list of ~190,000 potential gRNAs, targeting more than 40% of human exons. Scientists can search this list for their gene of interest. The Zhang lab has a similar online resource, which is home to a detailed background section on CRISPR technology, a discussion of the reagents in their system, and a tool for identifying potential targets in a variety of different species. The Joung lab also has a an online resource that includes a tool first developed for identifying potential Zinc Finger targets, then later modified to also identify TALEN array binding sites and is now also capable of identifying CRISPR target sites, all based on a user generated input sequence. There are other online, open access databases (such as http://crispr.u-psud.fr/) designed to help scientists find CRISPR targeting sites in a wide range of species and generate the appropriate crRNA sequence.
Figure 3: A simplified overview of the various engineered CRISPR technologies created from the bacterial Type II system. (A) A codon-optimized version of the Cas9 nuclease with a Nuclear Localization Signal (NLS) is expressed from an appropriate promoter. The Cas9 has been optimized for expression in various eukaryotic cell types including human. (B) A customizable DNA element that allows for the transcription of crRNA-tracrRNA fused hybrid RNA, often referred to as a guide RNA (gRNA). Generally the sequence to be targeted (protospacer) is inserted into the gRNA expression plasmid by synthesizing complementary oligos with the appropriate restriction site overhangs on the 5’ and 3’ ends. (C) Both the Cas9 expression plasmid and gRNA expression plasmid are then transfected into the target cell. In some cases a single plasmid containing two expression cassettes can be used. The single gRNA binds with and activates the codon-optimized Cas9 nuclease.
While the specifics differ between the various engineered CRISPR systems, the overall methodology is similar; a scientist interested in using CRISPR technology to target a DNA sequence (identified using one of the many available online tools) needs only to insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid. The gRNA expression plasmid contains the target sequence (~20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter and necessary elements for proper processing in eukaryotic cells. Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded DNA and then cloned into the gRNA expression plasmid. Double transfection of the gRNA expression plasmid and the Cas9 expression plasmid into your cells is all that is required.
Addgene is already the go-to source for the popular genome engineering kits utilizing TALEN technology and is now your source for the latest genomic engineering technology, CRISPRs. We strive to provide scientists all over the world with the opportunity and the means to share their work as easily as possible with the global community. Learn more about the CRISPR plasmids available at Addgene.
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