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Original: CRISPR/Cpf1

Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cpf1 is a DNA-editing technology analogous to the CRISPR/Cas9 system, characterized in 2015 by Feng Zhang‘s group from the Broad Institute and MIT. Cpf1 is a RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism found in Prevotella and Francisella bacteria is used to prevent genetic damage from viruses. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA, disabling viruses. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. CRISPR/Cpf1 could have multiple applications, including treatment of genetic illnesses and degenerative conditions.

Discovery
CRISPR/Cpf1 was found by searching a published database of bacterial genetic sequences for promising bits of DNA. Cpf1 appeared in many species. the ultimate Cpf1 endonuclease that was developed into a tool for genome editing was taken from one of 16 bacteria that harbored it. Two candidate enzymes from Acidaminococcus and Lachnospiraceae display efficient genome-editing activity in human cells.

It has been recently reported the discovery a smaller version of Cas9 in the bacterium Staphylococcus aureus, becoming a potential alternative to Cpf1.

Classification
The systems CRISPR/Cas are separated into two classes. Class 1 uses several Cas proteins together with the crRNA to build a functional endonuclease. On the other hand, Class 2 CRISPR systems use a single Cas protein with a crRNA. Cpf1 has been recently identified as a Class II, Type V CRISPR/Cas systems containing a 1,300 amino acid protein.

Structure
The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alfa-helical recognition lobe of Cas9.

Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique, being classified as Class 2, type V CRISPR system. The Cpf1 loci encode Cas1, Cas2, and Cas4 proteins more similar to types I and III than from type II systems. Database searches suggest the prevalence of Cpf1-family proteins in many bacterial species.

Functional Cpf1 doesn’t need the tracrRNA, therefore, only the crRNA is required. This has advantage for genome editing because Cpf1 is not only smaller than Cas9, but also it has a smaller sgRNA molecule (proximately half of the nucleotides number than Cas9).

Cpf1-crRNA complex cleave target DNA by identification of a T-rich protospacer-adjacent motif (PAM), in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpf1 introduces a sticky-end-like DNA double- stranded break of 4 or 5 nucleotides overhang.

Mechanism
The CRISPR/Cpf1 system consist of a Cpf1 enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA. CRISPR/Cpf1 systems activity has three stages:
 * 1) Adaptation: Cas1 and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array. .
 * 2) Formation of crRNAs: processing of pre-cr-RNAs producing of mature crRNAs to guide the Cas protein.
 * 3) Interference: the Cpf1 is bound to a crRNA to form a binary complex that will identify and cleave a target DNA sequence.

Ca9 vs. Cpf1
Cas9 requires two RNA molecules to cut DNA while Cpf1 needs one. The proteins also cut DNA at different places, offering researchers more options when selecting an editing site. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind ‘blunt’ ends. Cpf1 leaves one strand longer than the other, creating 'sticky' ends that are easier to work with. Cpf1 appears to be more able to insert new sequences at the cut site, compared to Cas9. Although the CRISPR/Cas9 system can efficiently disable genes, it is challenging to insert genes or generate a knock-in. Cpf1 lacks tracrRNA, utilizes a T-rich PAM and cleaves DNA via a staggered DNA DSB.

In summary, some important different between Cpf1 and Cas9 systems are:
 * Cpf1 recognizes a T-rich PAM. Cas9 recognizes G-rich PAM. Therefore, Cpf1 enables new targeting possibilities.
 * Cpf1 creates 4-5 nt long sticky ends, instead of blunt ends produced by Cas9; enhancing the efficiency of genetic insertions and specificity during NHEJ or HDR.
 * Cpf1 cuts target DNA further away from PAM, further away from the Cas9 cutting site. This enables new possibilities for cleaving the DNA.

Tools
Multiple aspects influence target efficiency and specificity when using the CRISPR system genome editing, including guide RNA design. Many design models for guide RNA has been suggested and an increasing number of tools are facilitating their optimized design. Computational tools for the general use of CRISPR systems includes SgRNA designer, CRISPR MultiTargeter, SSFinder and other software that facilitates target sequence finding and optimization.

Conflict of interest
Intellectual property concerns are keeping distant agricultural and drug companies from using CRISPR/Cas9. However, CRISPR/Cpf1 is new approached without legal matters, encouraging industries to invest and develop the Cpf1-based system.