A New Editing System

As described in a just-published paper in Cell by Zetsche et al (2015), Cpf1 is an endonuclease that is part of a CRISPR system somewhat similar to the Streptococcus pyogenes Cas9-based system that now is the tool of choice for genome editing and related manipulations but with notable and significant differences that potentially make it highly complementary to the SpCas9 system.

CRISPR Systems and Stages
Image Credit: CtSkennerton  Licensed under CC BY-SA 4.0 via Commons –

There are two or three classes of CRISPR systems that can be described as those with multiple proteins in the crRNA-containing effector complex and those with a single Cas protein with the crRNA in the effector complex.  The now familiar SpCas9 system is of the latter class (Class 2).

Zetche et al. characterize in some detail a CRISPR system from Francisella novicida that has only a single protein along with a cr-RNA in the effector complex.  The endonuclease, Cpf1 (CRISPR from Prevotella and Francisella 1), is quite distinct from Cas9 both in size (Cpf1 is 1300 aa while SpCas9 is 1629 aa) and sequence.

Zetche et al. go through a series of tests of the FnCpf1 system that are conceptually simple and you should consult the paper for the details of their findings but they make five major observations.

First, unlike the SpCas9 system, the Cpf1 system requires only a single crRNA in the effector complex.  No tracrRNA is required.  The practical implications of this is that gRNAs are short, only about 40 nt.

Francisella novicida is a Gram-negative bacteria with a Class 2 CRISPR system distinctly different from the Class 2 CRISPR system found in Streptoccus pyogenes.
Image from the CDC Public Health Image Library. Image credit: CDC/Larry Stauffer, Oregon State Public Health Laboratory (PHIL #1910), 2002

Second, Cpf1 cleavage results in 5′ overhangs of 5 nt.  These ‘sticky’ ends may allow for inserting sequences into the target site without relying on homology directed repair.  This too has practical implications since homology directed repair is effective only in dividing cells.

Third, Cpf1 cleavage occurs at the distal end of the gRNA, quite far from the ‘seed’ region of the gRNA.  SpCas9 cleaves a target sequence in the seed region of the gRNA so that cleaved and repaired targets are no longer substrates for the initial gRNA.  This is not necessarily the case with Cpf1 and this may have practical implications in the future.

Fourth, the Cpf1 system uses T-rich protospacer adjacent motifs (PAMs) – TTN.  SpCas9 PAM requirements limit target availability and there are Cas9 variants with altered PAM preferences but the Cpf1 system may be particularly useful for editing AT-rich genomes.

Cas9

The Cas9 Editing System

Fifth and finally, some of the Cpf1 systems tested seem to work well in human cells, compared with SpCas9.  Of the seven Cpf1 systems tested in human cells only those from Acidaminococcus (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) displayed active endonuclease activity in human cells. FnCpf1 actually did not show endonuclease activity in human cells.

Overall, this is an interesting and potentially important paper in that it describes a gene editing system that seems as facile as SpCas9 but with notably different characteristics, providing investigators with a new and unique tool by which they can accomplish their genome modifications.  It seems likely that insect biologists will be using this system shortly.

Zetsche, B., Gootenberg, Jonathan S., Abudayyeh, Omar O., Slaymaker, Ian M., Makarova, Kira S. et al.,  Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell: doi: http://dx.doi.org/10.1016/j.cell.2015.09.038.

 

 

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