CRISPR, or clustered regularly interspaced short palindromic repeats, describes a genetic motif in bacterial and archaeal genomes that encodes a suite of RNA tools used by a specific class of DNA-cutting proteins in the microbial immune system. Because of these proteins’ dependence on CRISPR, they’re called “CRISPR-associated,” or “Cas” for short.
Compared to other gene editing paradigms, CRISPR is inexpensive because it only requires the synthesis of a short segment of guide RNA and addition of a Cas enzyme: Cas9. And it’s simple to implement — just introduce a gRNA-Cas9 complex to DNA.
Cas9 was the first tool discovered from a larger toolbox of CRISPR-associated proteins. This article will summarize some CRISPR tools, including descriptions of the specialized purposes that each variant befits.
Cas9 is a DNA-cutting enzyme that couples with an RNA molecule called a guide RNA (gRNA) to break apart DNA wherever there is a nucleotide sequence matching the complement of the gRNA sequence.
The nucleotide sequence of the gRNA template matches the target site, guiding the Cas9 endonuclease to this position. By simply synthesizing a custom-designed gRNA sequence, researchers can accomplish precision gene editing at any site of interest.
Cas9 exists in many bacterial species, and differs slightly between species. One of the main differences between species is in the sequence of a specific motif that has to occur in the targeted sequence for Cas9 to make a cut. The form of Cas9 found in the bacteria Streptococcus pyogenes is the most thoroughly characterized variant by virtue of its target motif simply being “NGG”, making it the most versatile Cas9 discovered to date.
Cas9 is an enzyme with multiple functional domains distributed over two lobes. Some of these domains identify the DNA target, while other sites act as scissors, cutting the DNA target. With targeted mutations, the nuclease domains can be deactivated, effectively blunting these scissors while maintaining Cas9’s DNA targeting activity. Researchers can then leverage CRISPR’s precision and simple implementation for purposes other than genetic engineering.
This deactivated version of Cas9, known as dCas9, can be fused to other non-specific DNA-acting proteins, allowing them to be targeted to any sequence of interest. Some researchers have used dCas9 as a mapping tool, charting the distribution of genes on a chromosome. Other researchers use dCas9 to target genes with transcription factors in order to study epigenetic influences on the genome.
dCas9 has both DNA cleaving domains deleted. Alternatively, by only perturbing a single domain, Cas9 becomes a highly precise DNA nicking enzyme. Cas9 that is altered to break only a single strand is thus known as Cas9n.
Cas9n is useful for controlling the DNA repair process. The cell has nick repairing pathways that are independent from those activated by double stranded break damage. However, both nicks and double stranded breaks have their own HDR pathways. By using Cas9n, researchers can bias their experiment toward HDR, improving the efficiency of genetic knock-ins and ensuring any unwanted effects from NHEJ can be avoided.
Unlike the Cas9 variants described previously, which were invented by deliberately modifying the Cas9 protein, Cpf1 is a distinct protein with a similar function to Cas9. Cpf1 belongs to the same family of CRISPR-associated endonucleases as Cas9 and as such, shares many of Cas9’s functions, like its use of guide RNA to target a region of DNA as well as its double-strand severing ability. But Cpf1 performs these functions slightly differently than Cas9.
Some of Cpf1’s variations make it preferable to Cas9. One advantage is that Cpf1’s gRNA is a simpler, shorter structure, as well as easier to engineer than the gRNA for Cas9. A second advantage is that Cpf1 has a target motif of “TTN.” Whereas the key nucleotide sequence motif that Cas9 recognizes grants access to many regions of a typical genome, there are inevitably some regions that can’t be accessed because of a lack of adjacent di-guanines. Thus, Cpf1 opens up whole new DNA regions to CRISPR that weren’t previously accessible.
C2c2 is a CRISPR endonuclease that operates in a manner like Cas9 and Cpf1, but with a twist. Instead of precisely breaking targeted DNA sequences, C2c2 targets RNA sequences.
Because of how C2c2 ignores DNA in favor of RNA, it is useful in scenarios where transient effects are the goal. One of CRISPR-Cas9’s most appealing qualities is that it can introduce permanent and heritable edits into DNA. However, in many instances, permanent changes are not the desired outcome. With C2c2, researchers can transiently alter gene expression by cleaving RNA transcribed from a target gene.
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