CRISPR-Cas9 is the most well-known gene editing tool out there, which combines a guide RNA (molecular GPS) with a Cas endonuclease (molecular scissors) to finely target and manipulate genes.

However, Cas9 is just one tool of many, ranging from Cas1 to Cas14. Let's dive into all the rest.

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Cas1—the Hard Drive

Cas1 is a record keeper, which works in complex with Cas2 to capture invading bacteriophage DNA. Remember that CRISPR functions as a bacterial immune system to protect against phages.

Cas1 acts like a molecular ruler, measuring out a piece of DNA of the correct length. After capturing the invading DNA, the Cas1-Cas2 complex bends and snaps this DNA into the CRISPR array as a new "spacer." Each spacer acts as a memory of a specific phage infection for future reference.

Cas2—the Hard Drive, part 2

As mentioned, Cas2 and Cas1 work in complex. Two copies of Cas2 join with four copies of Cas1 to create a DNA capture complex. This Cas1-Cas2 complex roams the cell, searching for invading bacteriophage DNA.

Cas3—the Shredder

Once Cascade identifies a double-stranded DNA (dsDNA) target, it recruits Cas3, which uses ATP to unwind invasive bacteriophage DNA and wreck it to bits.

"CRISPR-Cas3 can efficiently erase long stretches of DNA from a targeted site in the human genome, a capability not easily attainable in more traditional CRISPR-Cas9 systems."

Cas4—the Reviewer

Cas4 recognizes the correct PAM and nucleotide motifs of protospacers and determines the spacer length as well as its orientation. This step ensures that only usable new spacers are added to the CRISPR array.

Cas proteins use the PAM sequence to find their target, and don't target DNA not flanked by a PAM, making PAM a "safeguard" to prevent Cas proteins from cutting the bacterium's own DNA. Cas4 makes sure that only sequences flanked by a PAM are inserted into a bacterium's DNA.

In simpler terms, "only when Cas4 was present, snippets of invading DNA were inserted in the genome of the bacterium that actually conferred virus resistance to the bacterial cell."

Cas5—the Stabilizer

Cas5 binds to one end of CRISPR RNA (crRNA) in Cascade, capping the end of the complex. Cas5 also helps to "process or stabilize pre-crRNA into individual crRNA units." In complex with Cas6, it is used for optimal crRNA processing and stability.

Cas6—the Generator

Cas6 is needed to generate crRNAs in CRISPR-Cas I and III systems. Remember that a guide RNA is made of a tracrRNA and crRNA, where the crRNA is complementary to the target DNA.

Cas7—the Backbone

A series of six Cas7 proteins form the Cascade backbone. Each protein loops around the CRISPR RNA (crRNA). These proteins also help bind target DNA.

Cas8—the Security

In Cascade, Cas8 identifies bacteriophage DNA by checking for a PAM sequence. It also helps unwind the target DNA and recruits Cas3 to destroy it.

Cas9—the Editor

Cas9 is a DNA binding and cutting enzyme directed by a guide RNA. One part of the RNA acts like a handle for the protein to grab. Remaining nucleotides form base pairs with complementary DNA. Once a target is bound, Cas9 cuts each strand.

Cas10—the Phage Editor

Cas10 is a Type III-A CRISPR-Cas system native to Staphylococcus epidermidis, and can be used as a tool for phage genetic engineering.

Cas11—the Cascade Belly

Three Cas11 proteins form the belly of the Cmr/Csm Cascade complex, orienting the target RNA, facilitating base-pairing with the CRISPR RNA (crRNA) and cleavage by backbone subunits.

Cas12—the Cautious Editor

Cas12 (also known as Cpf1) uses a CRISPR RNA (crRNA) guide to identify matching DNA that it binds and cuts. Unlike Cas9, after Cas12 finds its target, it also starts cutting nearby single-stranded DNA. Cas13 is similar, but it cuts RNA.

The cutting mechanism is staggered. Recent discoveries show that Cas12a can indiscriminately chop up ssDNA once activated by a target DNA molecule matching its spacer sequence. This makes Cas12a a strong tool for detecting tiny amounts of target DNA in a mixture.

Studies suggest the Cas12a is a more careful editor and is more precise than its cousin Cas9.

Additional reading can be found here.

Cas13—the RNA Outlier

Cas13 uses a CRISPR RNA (crRNA) guide that directs it to complementary pieces of single-stranded RNA, making it an outlier in the CRISPR world.

If the target is a match, Cas13 cuts it. Once activated by finding its target, the enzyme starts cutting nearby RNA. Cas12 is similar, but its targets (both complementary and collateral) are DNA. Cas13 can be used to detect very small amounts of RNA and diagnose infection or disease.

Cas14—the Cas9 Ancestor

Cas14 encodes for a small Cas protein, roughly half the size of other Cas proteins in class 2 CRISPR systems. Unlike other Cas enzymes, Cas14 is not found in bacteria, but in Archaea, suggesting that Cas14 is a more primitive version of the more complex Cas9 and Cas12 systems.

Cas14 can bind and cleave a targeted sequence of ssDNA, and unlike Cas9, does not need a PAM sequence. Since it doesn't cut dsDNA, Cas14 isn't a good genome-editing tool candidate, but it could be used for diagnostics, by now enabling the detection of ssDNA.

Understanding the Different CRISPR Systems

We've explored fourteen CRISPR-associated Cas nucleases, which are categorized by their functionality and "effector molecules." The main classes are Class 1 and Class 2.

Class 1 effectors have multiple subunits while Class 2 effectors are single large proteins.

This table by Synthego summarizes the Class 1 and Class 2 differences: