This past weekend at the Pilgrimage music festival in
Franklin, TN I had the pleasure of seeing Weezer rock out, then I walked a few
hundred yards to another stage to watch Wilco do the same. These bands each had their own stage,
but in the CRISPR world, Cas9 is a superstar that now has to share the stage
with a newcomer: Cpf1. (Yeah, I know that's a goofy setup but it
really was a good festival and it was on my mind. Now on to the science.)
Last Friday Feng Zhang’s group published a paper in Cell
that immediately grabbed a lot of attention, and rightly so. They reveal that the Cpf1 class of
CRISPR effector proteins may be an attractive alternative to Cas9. Although Cpf1 has many
similarities to Cas9, it has some significant differences that are very
interesting – and could lead to improved efficiencies for some types of gene
editing.
Here is my summary…First, they reviewed some background on
CRISPR systems in bacteria to cover the basis of the study. There are two major classes of
CRISPR systems based mainly on the proteins involved; the cleavage effectors of
class 1 are complexes of multiple proteins, while the class 2 effectors are
single proteins like Cas9. Within
class 2 there are two subtypes of systems: those with Cas9, and another that has Cpf1. Cpf1 means “CRISPR from Prevotella and Francisella 1”. Some
bacterial species carry both Cas9-CRISPR and Cpf1-CRISPR loci in their
genomes. Like Cas9, Cpf1 has
RuvC-like DNA cleavage domains but it lacks some of the other domain and
neighbor-gene features of Cas9, so it’s clearly distinct in its evolution. It’s a largish protein of
~1300 amino acids, similar in size to Cas9.
They picked the Cpf1-CRISPR gene system of Francisella novicida strain U112 to
study first since there were clear homologies of Cpf1-CRISPR spacer sequences
to various prophage in this species – further suggesting that Cpf1 is important
in bacterial immunity and so it’s well adapted to slice and dice target
DNAs. (Sidebar: what’s F. novicida? A pretty rare human
pathogen, originally isolated from the Great Salt Lake in Utah. It’s related to the better known bug F. tularensis which is one of the most
infectious pathogens known.)
By transferring the F.
novicida Cpf1-CRISPR gene locus into E. coli they quickly established that
it prefers a “TTN” PAM motif that
is located 5’ to its protospacer target – not 3’, as per Cas9. So right away it’s distinct in having a
PAM that isn’t G-rich and is on the opposite side of the protospacer.
Like Cas9, Cpf1 binds a crRNA that carries the protospacer
sequence for base-pairing the target.
But for me the biggest surprise in the paper is that unlike Cas9, Cpf1
does not require a separate tracrRNA – in fact, there’s no sign of a tracrRNA
gene at the Cpf1-CRISPR locus.
Thus, Cpf1 merely needs a cRNA that is about 43 bases long –of which 24
nt is protospacer and 19 nt is the constitutive direct repeat sequence. This is very different than Cas9
– even by fusing the crRNA and tracrRNA, the single RNA that Cas9 needs is still ~100
nt long.
Furthermore, the Cpf1 crRNA does not have the long stemloop
structure that is typical of RNAseIII-mediated processing to cut it out of its
primary transcript. It has a
much shorter stemloop that is required for Cpf1 activity, however. But surprisingly, Cpf1 itself is apparently directly
responsible for cleaving the 43-base cRNAs apart from the primary transcript in
the first place! This isn’t conclusively proven yet, but is pretty likely based
on their experiments.
Next, two more surprises comes from the cleavage sites on
the target DNA. The cut sites are
staggered by about 5 bases. This
should create “sticky overhangs” that might
be exploitable to enable gene editing via NHEJ-mediated-ligation of DNA
fragments with matching ends.
And, the cut sites are in the 3’ end of the protospacer, distal to the
5’ end where the PAM is.
The cut positions usually follow the 18th base on the
protospacer strand and the 23rd base on the complementary strand
(the one that pairs to the crRNA).
They tested if they could inactivate the DNA cleavage
domains via homologous mutations in codons known to do this in Cas9. However, the resulting Cpf1 mutants
don’t have “nickase” activity – they can’t cut either strand. So it’s not clear that Cpf1
nickases can be made and in fact the authors suggest that the cleavage might
require some sort of dimerization.
I can’t visualize how that would work yet but I’m sure it will be
figured out in the near future…
Base substitution experiments then showed that, as per Cas9
CRISPRs, there is a “seed” region close to the PAM in which single base
substitutions completely prevent cleavage activity. Therefore, unlike the Cas9 CRISPR target the cleavage
sites and the seed region do not
overlap. This immediately suggests
a potential improvement over Cas9 in mammalian HDR-mediated repair
efficiency. This is
because any initial cleavage events that might lead to “simple” NHEJ indels
might still be substrates for
cleavage - and thus allowing
additional chances for HDR-mediated edting to occur. With Cas9, an indel mutation will almost always disrupt the
target seeds and then it’s game over for HDR.
Finally, they did the important work to screen various Cpf1
proteins from different bacterial species to see if any would actually work in
mammalian cells. This is because
that despite codon optimization and attachment of nuclear localization signals,
most of these bacterial proteins just don’t work right when you put them inside
human or mouse cells. Therefore
they tested 16 different Cpf1 proteins.
Of these, for seven proteins they could identify PAM signatures using
their E. coli assay. They all had similar
T-rich PAMs.
Of these seven proteins, only two worked well in human HEK293 cells – AbCpf1, from an Acidaminococcus, and LbCpf1, which is
from a Lachnospiraceae; interestingly
these are apparently both anaerobic bacteria sometimes found in mammalian intestines. Anyway these can both generate indels at
specific targets in human cells at a rate similar to Cas9 – typically, 10-20%
Surveyor assay numbers were observed, when they tested HEK cells following simple
transfections.
Bottom line: Cpf1 may be the real deal as a serious competitor
for Cas9. Is Cas9 suddenly
obsolete? Hardly. First, we don't yet know if Cpf1 is as specific as Cas9 – though there is every
reason to think it may be. So the off-target effects need to
be carefully measured. Second, we don’t know how widespread the targeting
efficiencies will be across sites (although the initial tests seem very
promising). Third, although Cpf1
may be better than Cas9 for mediating insertions of DNA, it’s not yet been
shown if that is true. However it may have some nice advantages over Cas9, not the least of which is that its guide RNA is only 43 bases long. It will thus be feasible to purchase directly synthesized guide RNAs for Cpf1, perhaps with chemical modifications to enhance stability.
Probably, Cpf1 and Cas9 will both be in the spotlight for a
long while to come. Look for more
Cpf1 papers to start coming out very soon and for Cpf1 plasmids to appear in
Addgene. Happy CRISPRing,
everyone.
