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The Development and Application of CRISPR-Cpf1

Genome editing is slowly causing, or has perhaps already caused, a paradigm shift in the world of agriculture and in plant genomics in general. The ability to precisely and easily edit genes has never been as widespread before as it is now. The technology is causing a momentous shift towards using genome editing to not only validate gene function but also to create better crop varieties for the sustenance of a growing human population.

CRISPR: the specifics

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is the adaptive immune system that is present in Prokaryotes and Archaea. The main function of this system is to protect the microbe from virus-based attacks. The basic premise involves incorporating small portions of the viral DNA in the bacterial genome during an infection and then using these fragments as recognition motifs during subsequent attacks by the same virus. The viral DNA fragment is transcribed and the RNA binds to the incoming viral DNA. Then an endonuclease, coded by the bacterial immune system, goes to the location of binding and creates a double-stranded break in the viral DNA, thereby negating the chance of an infection. The binding of the target location is also dependent on the existence of a protospacer adjacent motif or PAM.

This entire system was discovered in the dairy industry when scientists began to see that certain starter cultures managed to resist bacteriophage and viral infections. While there are a host of proteins that are involved in the entire defense mechanism, scientists soon figured out that if the target DNA was modified and the correct motifs were selected for identification of the target, then the endonuclease can be virtually be used to generate double-stranded breaks (DSBs) in any genome of any organism. This led to the rise of application of CRISPR as a tool for genome editing. It is fast, precise, and easy to modulate using a single guide RNA, which can be modulated along with a CRISPR effector.

CRISPR from Prevotella and Francisella 1

The initial research with CRISPR focused on protein 9 or Cas9. Cas9 is the RNA-guided DNA endonuclease, originally isolated from Streptococcus pyogenes, that target the GC rich regions of a genome. While this was an important component in the entire genome editing tool, it had its own share of limitations, including the ability to target the AT-rich regions of the genome and having PAM limitations. But in 2015, with concentrated efforts in Broad Institute, MIT and other universities, a new CRISPR effector was isolated for ease of editing the genome (Zetsche et al., 2017). This was named as CRISPR from Prevotella and Francisella 1 (or Cpf1 for short).

The major defining features of Cpf1 that separate it from Cas9, is the fact that it has an AT-rich PAM and can target the AT-rich regions of the genome. Moreover, its structure differs from Cas9 because it does not have an HNH domain, but rather three RuvC domains. These features mean that Cpf1 is the standard choice of nuclease in genome editing.

In addition, while multiple proteins are required during the formation of crRNAs, Cpf1 can process its own crRNAs, which makes it really flexible and adaptable. Moreover, while Cas9 requires a tracrRNA to pair with the crRNA to make it functional, a single crRNA is enough to make Cpf1 functional. What this translates into in terms of synthesizing or cloning this set up is the possibility of shorter scaffold for designing the single guide RNA. In addition, while Cas9 introduces a blunt-ended double-stranded break, Cpf1 creates 4-5 nucleotide long, sticky ends. This is hugely advantageous in terms of precise insertion for fragments of nucleotides during the DNA repair, mechanisms of Non Homologous End Joining, or Homology Directed Repair. Lastly, while Cas9 induces the DSB near the PAM region, Cpf1 causes the cut to be away from the PAM, which means that new edits or cuts can be made during every round of interaction.

Multiple Targets, Single Nuclease

While there has been consistent development in the targeting abilities of Cpf1, it was initially used to target single locations in the genome. It was soon figured out that if the structure or the array of the arrangement of the Cpf1, crRNA, and the nuclease that is present inside the bacteria is taken as a basis and a similar array with pre-crRNA sequences, matching the target locations of one’s choice is designed; then it would be possible to target multiple locations in the genome at the same time. This would generate multiple edits in a single generation and increase our ability to produce more targeted insertions, gene validations, and interactions. This strategy, known as using the CPf1 array, is now being widely employed to practice multiple targeting or multiplexing via Cpf1. Since Cpf1 can process its own crRNA (while Cas9 cannot), all that is needed is a long DNA fragment array, harboring the target sequences of your choice, separated by the recognition site for the Cpf1 to process the crRNA. Then the Cpf1 can process the crRNA in vivo and thus target multiple locations.

Employing the technology

Our group in IRRI is attempting to use both Cpf1 single targeting, working on a stomatal developmental gene (Yin et al., 2017), and Cpf1 array-based multiplexing systems for application in photosynthesis based research in rice. While the technology is still in its developmental phases, there are tremendous opportunities for it to burgeon into the genome editing tool of choice.

To learn more about the development and application of Cpf1, listen to Anindya Bandyopadhyay speak at the Plant Genomics and Gene Editing Congress: Asia.  Download the agenda here.

Authors

Anindya Bandyopadhyay is a molecular biologist and is the Group Leader for genome editing at IRRI’s C4 rice centre and Dr. Shahid M. Mukhtar is an associate professor at the UAB College of Arts and Sciences, Birmingham, Alabama.

References
  • Biswal, Bandyopadhyay et al. (2017) CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice. Plant cell reports. 36(5):745-57.
  • Zetsche, Gootenberg et al. (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell.;163(3):759-71.

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