CRISPR, which stands for Clustered regularly interspaced short palindromic repeats, are naturally occurring repeated DNA sequence present in bacteria and archaea family.
They work together with CRISPR associated (Cas) genes and enzymes to act as a defense system against invading phage or plasmid DNA. Cas9 (CRISPR associated protein 9) is RNA-guided DNA endonuclease enzyme found in type II CRISPR system. It is most commonly found in Streptococcus pyogenes as a defense system.
Between the palindromic repeats OF CRISPR short variable sequences, derived from exogenous DNA targets, is present which is known as protospacers. Together they form the CRISPR RNA (crRNA) array.
Protospacer is always associated with a protospacer adjacent motif (PAM), which is different in different CRISPR system. Presently there are three CRISPR/Cas systems being discovered yet from which type II is the most common one.
Essential components of CRISPR/Cas9 system
There are basically two main components of Cas9 system for gene editing which include:
- Guide RNA or gRNA: It directs the Cas9 enzyme to a precise genomic locus in targeted double stranded DNA molecule.
- Cas9 endonuclease: An enzyme which is used to snip target DNA.
Hence genome editing is achieved when these two components are successfully expressed in a living cell.
Mechanism of editing technology
After the formation of double strand breaks (DSB) genome editing is done by two different mechanisms. They include:
- Non-homologous end joining
- Homology directed repair
Non-homologous end joining
This type of DNA repair occurs in the absence of a homologous DNA template. In the absence of DNA template DSB can be ligated resulting into insertion/deletion (indel) mutations. NHEJ can be used to result in gene knockouts, as indels and can lead to frame-shift mutations and premature stop codons consequently this is an error prone mechanism.
Homology directed repair
This type of DNA repair occurs in the presence of a synthetic repair template. The repair template can be a conventional double-stranded DNA targeting constructs with homology arms flanking the insertion sequence, or single-stranded DNA oligonucleotides (ssODNs).
The ssODNs are more effective in causing genetic variations. HDR can be used to generate precise, defined modifications at a target locus therefore this mechanism has high fidelity as compared to NHEJ.
Modified forms of CRISPR/Cas9 system
Now days other than the wild-type Cas9 two other variants of Cas9 nuclease are also being used. They include Cas9 nicakse and Cas9 double mutant.
This mutant form of Cas9 cleaves only one DNA strand. DNA repairs are done by the high-fidelity HDR pathway only. For genome editing two gRNAs are used which cleaves separate single strands in close proximity. This variant was developed to overcome off target effects.
Cas9 Double Mutant
This is a nuclease deficient Cas9 variant. It binds with target genome but cannot cause cleavage so it does not carry out genetic modification. It can be fused with different effect or domains and can be used as gene silencing, activation tool or visualization tool.
The efficiency in the delivery of Cas9 enzyme and gRNA affects the success of genome editing. There are two types of delivery system that are being most commonly used. They include:
- Viral delivery
- Non-viral delivery
Viral delivery involves the use of an intermediate vector for the transfer of gRNA and Cas9 into the targeted organism. For this purpose AAV (adeno associated viral vectors) have been successful used. The benefit in using them is that they do not cause pathogenicity and can affect both dividing and non-dividing cell.
But it has a drawback that is it has small genome size, but this can be overcome by using smaller sized Cas9 enzyme. Other vectors that can be used include lentiviral vectors and adenoviral vectors, which can accommodate large payloads.
Non-viral gene delivery
Non-viral delivery involves different chemical and physical methods. The most efficiently used method is cationic lipid-mediated transfection. It involves Cas9 protein delivery through cationic lipids complexed with polyanionic sgRNA using lipid transfection agents.
Other non-viral delivery methods include calcium phosphate transfection, hydrodynamic tail vein injections of saline DNA solutions, polymer nano-particles, electroporation etc.
Advantages of using CRISPR/Cas9
CRISPR/Cas9 technology has a lot of benefits over previously used tools. It is easy, cheaper and more reliable. It allows scientists to make use of in-house kits or commercially available kits, only what they have to do is to choose the location of DNA double strand break and then order an oligonucleotide.
Following are some of the benefits of using CRISPR/Cas9 as genome editing tool:
Ease of customization: Cas9 can be used to easily target any DNA sequence simply purchasing a pair of oligos encoding the 20-nt guide sequence.
Cleavage pattern: Cas9 can be used to cleave at any desired loci. Furthermore modifications can be made to cut only one and even none of the DNA strand.
Editing efficiency: Because of ease of targeting Cas9 can be used to target many genomic loci simultaneously. For this a combination of sgRNA can be delivered to the cell of interest at the same time.