International MPN News, Science & Opinion

Cpf1 – A faster, smaller, better CRISPR enzyme?

There was no way to get ready for Feng Zhang’s opening night performance at the CSHL gene editing meeting.

The “CRISPR/Cas Revolution” meeting at Cold Spring Harbor Laboratory last week was about to witness a bombshell introduction of a new CRISPR enzyme : CRISPR/Cpf1.

Feng Zhang, MIT

Feng Zhang, MIT

Thursday evening, September 24, Feng Zhang delivered his rapid fire presentation, profusely illustrated with projected slides.   The assembled scientists, post docs, CRISPR specialists, and academics expected to hear a talk billed as “Genome editing using CRISPR Cas-9.”  Instead what they witnessed was an historic, electric moment in the brief tumultuous history of gene editing.

On-line media coverage of Cpf1 came pouring out the next day. “CRISPR-Cpf1 offers simpler approach to editing DNA; technology could disrupt scientific and commercial landscape,” headlined the Broad Institute in the MIT News. “New CRISPR/Cpf1 Genome Editing System Makes Waves at CSHL Meeting,” announced genomeweb.  And from  “Alternative CRISPR system could improve genome editing.” “The War of Genome editing just got a lot more interesting,” Wired.

It had to take a certain amount of chutzpah for Feng Zhang to unveil CRISPR/Cpf1 at a genome editing conference convened by Jennifer Doudna and her colleagues.  Given the stormy intellectual property history in which Jennifer Doudna and Feng Zhang have been key players over the past three years — and the dominant role CRISPR/Cas9 has played ever since —  introducing his competing Cpf1 enzyme in this venue would surely create a shockwave.

Cpf1 is an entirely different approach to CRISPR-based gene editing.

That there would eventually be an alternative or many alternatives to CRISPR/Cas9 was a given. The CRISPR sequence, a repeated pattern within the DNA of bacteria serving immune function by storing invading viral DNA sequences for future targeting, is associated with cutting enzymes. Cas9 is one of the enzymes associated with Streptococcus pyogenes, the RNA-guided endonuclease presented by Doudna and Charpentier in 2012. The Cpf1 enzyme is smaller, produces an overhang cut versus the blunt Cas9 DNA cut and requires only one RNA for targeting.

Possibly of greater significance, Cpf1 recognizes a different protospacer adjacent motif (PAM). This is the landing site for editing and the PAM sequence is not on the end of the spacer  but the beginning, located 5′ to the DNA target site.   Whether or not all this makes a difference in genetic research remains to be seen, but clearly there is a new kid on the block.

Feng opened his presentation outlining the promise of  better CRISPR system.  Cas9 belongs to the Class II system made up of a single proteinCpf1 staggered cut with multiple subunits for RNA binding and DNA recognition and subsequent cleavage function.  In Class 1 CRISPR systems multiple proteins act as a complex system to bind to the RNA recognize the DNA and carry out subsequent instructions.

“This, said Feng, “led us to explore other Class 1 enzymes – leading to Cpf1. His lab studied the  CRISPR from the Prevotella and Francisella (PreFran) CRISPR subtype.

His lab developed a candidate from a published resource of multiple sequence alignments database, derived candidates and then started testing for PAM sequences.   Listening to Feng describe the process conveyed some idea of the sheer persistence and analysis required to unearth this enzyme.

“We cloned out the PreFran locus and introduced an expression vector via an e coli with a library of sequences, 7 or 8 before the spacer sequence or 7 or 8 bases after the spacer sequence.  So we can see there are specific plasmids from this pool that are depleted.  By looking at the depleted plasmids we could infer a specific motif that is probably the PAM sequence.”

And then came disappointment.

He found 40 or so Cpf1 proteins but “didn’t observe promising results when tested in human cells.”

The next steps outlined by Feng in a flurry of slides was to narrow his search down to 16 orthologs with aligned repeats, test the PAM sequences, test lysates for indel (insertion/deletion) creation to see if his candidates were capable of genome editing and derived two likely candidates. “Out of 16 Cpf1-family proteins,” said Feng, ” we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. ” The full details in Cell.the next day.

Zhang concludes: “We are committed to making the CRISPR-Cpf1 technology widely accessible. Our goal is to develop tools that can accelerate research and eventually lead to new therapeutic applications. We see much more to come, even beyond Cpf1 and Cas9, with other enzymes that may be repurposed for further genome editing advances.”

Note On plasmids and AddGene

Plasmids are the Swiss Army knife in the molecular biologist’s tool kit.  These little bits of bacterial DNA — also called vectors —  can be engineered to perform multiple CRISPR gene editing functions — CUT a double strand of DNA or NICK a single strand, shut down a gene or turbocharge its expression.  Plasmids are fragments of double-stranded DNA.Skillsaw blade They can replicate independent of the chromosomal DNA. They’re small, easy to work with, stable and versatile. Herbert Boyer back in 1972 discovered an E.coli enzyme, EcoRI, could cut DNA at specific sites. Splicing together fragments of different plasmids the resulting recombinant DNA now coded for a specific function could be cloned and rapidly mass produced by the bacterium.

AddGene, a non-profit repository for plasmids,PlasmidMap.png is  kind of Joy of Cooking for genetic engineers, containing descriptions of basic processes, recipes, and ingredients  to perform CRISPR gene editing.     Its gene editing guide is a short course for beginners, well worth checking out.  As might be expected,  both the  Doudna and Zhang labs are heavy contributors to AddGene.


Take me back to the Contents

© 2015, MPNforum. All rights reserved under Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License



%d bloggers like this: