Table 1.

Summary of Type 2 CRISPR system tools.

CRISPR System	Identification	Formation and Parts	DNA Binding	Action Upon Binding DNA	Ref.
Natural	A target site in the bacteriophage genome is identified through a Cas1–Cas2 complex which binds the invading viral DNA and takes a region that it then integrates into the CRISPR array, ensuring a PAM sequence adjacent to this site is present before choosing this spacer. Different subtypes of type II systems require other proteins, such as II-A needs Cas9 and Csn2 and II-B needs Cas4.	Upon future bacteriophage infection, the CRISPR array is transcribed, transcribing the spacers from past infections. These then complex with Cas9 and the tracrRNA, forming the CRISPR complex.	The correct spacer for the current infection and Cas9 will then bind the viral DNA at the site. The spacer is specific and complementary to Cas9, recognizing the PAM sequence, as is required for binding.	The Cas9 protein, specifically the RuvC and HnH domains, cleave the double-stranded DNA genome of the bacteriophage, stopping the infection.	[33,36,38]
Engineered	A disease is identified through the phenotype shown with the disease before studying the genome to find an appropriate target gene and a target site within this gene with an adjacent PAM sequence which a guide RNA can be designed against for targeting.	The tracrRNA and spacer are merged to form a guide RNA. This is then put in a vector with the Cas9 gene, which is delivered to cells and the guide RNA and Cas9 are expressed, forming the CRISPR complex.	The guide RNA is designed to be complementary to the target DNA, which is causing a disease, so it will bind at the target site with Cas9, which recognizes the adjacent PAM sequence, allowing binding so that Cas9 can then cleave the DNA.	The RuvC and HnH domains of Cas9 cleave double-stranded DNA, which is then repaired either through NHEJ or HDR. HDR can be used with an exogenous template to insert a sequence into the genome and correct mutations.	[38,46,48,50]
Transcriptional editors	The aberrant phenotype shown is used to identify the disease before analyzing the genome to find the gene responsible, and a guide RNA is then designed against a site within this gene with an adjacent PAM sequence to change the gene expression through epigenetic modification.	A guide RNA is put in a vector with s-gene coding for the deactivated Cas9 fused to a transcriptional activator or repressor. This is then expressed in the cell, forming the complex.	The guide RNA and Cas9 bind the DNA through Cas9, recognizing the PAM sequence allowing the guide RNA to bind the region it is targeted against. Here Cas9 binds, not for cleavage, as it is deactivated, but for the attached transcriptional control genes to function.	Upon binding the DNA, the transcriptional control gene fused to Cas9 will perform its function by either introducing or removing an epigenetic modification such as a methyl group, which will increase or decrease the gene expression, depending on the modification made.	[38,46,60]
Base editors	The phenotype shown is used to identify the disease before analyzing the genome to find the mutation responsible, and a guide RNA is designed against the mutated site. An adjacent PAM sequence is still required for binding, so some mutated sites may not be possible to target if there is no PAM sequence present. These can only correct transition mutations, not transversion mutations or indels.	Base editors contain a guide RNA with a partially inactivated Cas9 (Cas9 nickase) to cleave one strand. They also have a base editor—either a cytosine or adenosine deaminase fused tthis Cas9 nickase. This is all included in a vector which is then expressed in the cell where the different elements will come together to form the complex.	The guide RNA binds as it does in engineered systems with Cas9 nickase recognizing the PAM site and the guide RNA binding to its target region. Cas9 nickase will only nick one strand whilst the deaminase associated with it acts on the other strand.	Either cytosine or adenine is deaminated depending on which deaminase is being used, to go to uracil or guanine, respectively. Cas9 nickase will nick the other strand, which are then repaired to match the changed base.	[38,46,60]
Prime editors	Identification is carried out in the same way as for base editors, including ensuring the mutated site has an adjacent PAM sequence, but when the guide RNA sequence is made, a correct sequence to be copied in, in place of the mutated sequence, is included. This method means both transition and transversion mutations can be corrected. Indels can be repaired using this method through the inclusion or removal of a base/bases in the prime editing guide RNA that has/have been inserted or deleted in the mutated genome.	Prime editors contain Cas9 nickase, which will cleave one strand fused to reverse transcriptase and a prime editing guide RNA made up of a normal guide RNA, a reverse transcriptase primer binding site, and a sequence to be copied in. This is all put in a vector which is expressed in cells forming the CRISPR complex.	Cas9 nickase recognizes the PAM sequence with the prime editing guide RNA binding the target sequence. Reverse transcriptase binds with Cas9 as it is fused to it.	Firstly, Cas9 nickase nicks one strand. The prime editing guide RNA used in prime editors is longer, containing a sequence that is copied into the genome by reverse transcriptase to correct a mutation, with the original mutated strand replaced by this new edited strand. The other strand is then cleaved and repaired to match the edited strand.	[38,46,60,62]