DELETION MUTATIONS
The present invention relates to a process for removing genetic material from a bacterial
cell, specifically producing deletions in bacterial genomes or eliminating endogenous
bacterial plasmids. In particular, the process relates to the transformation of bacterial
cells with one or more Deletion Vectors, wherein the Deletion Vectors are capable of
directing production of two or more crRNAs which target two or more PAM/Protospacers
within the genomes of the bacteria within the population or within endogenous bacterial
plasmids. The bacterial DNA is thereby cleaved in two or more places, and the resulting
linearised fragments will be susceptible to degradation by endogenous bacterial
mechanisms. If this occurs within the bacterial genome, the two cleaved ends of the
major fragment may rejoin, while the minor linear fragment (being shorter) is degraded.
If a bacterial plasmid is targeted, even the major fragment will be relatively small and
therefore it is likely to be degraded rather than rejoined, resulting in removal of the
plasmid from the cell.
Solvent-producing Clostridia have been used since the 1910s for the industrial
production of acetone, butanol and ethanol. During the 1950s, the establishment of
more efficient petrochemical techniques to synthesise these solvents lead to the
abandonment of such large-scale bacterial fermentations. However, in the present
environment, with increasing pressure for the development of chemicals using
sustainable and renewable processes, the interest in clostridial fermentations for the
production of solvents is being renewed. This has also been helped by advancements in
the biological understanding of these solventogenic Clostridia, with the sequencing of
several genomes and the use of RNA sequencing and transcriptomics. These areas of
research have opened up the possibility of engineering new strains which are capable
of over-producing butanol, or removing production of competing by-products, further
improving the economics of solventogenic fermentations.
In order to take advantage of this influx of genomic information, there remains a need
for quick and effective methods for generating recombinant clostridial and other
bacterial strains to facilitate research and understanding, which will lead to the
generation of commercially-relevant strains.
It has been traditionally very difficult to generate recombinant clostridial strains. Low
transformation efficiencies in combination with low recombination efficiencies have
hampered efforts to make stable recombinant strains exhibiting improved solventrelated
phenotypes. Over the past few years, technology has been developed that
allows insertional inactivation of genes through use of Type I I introns, e.g. Targetron
(Sigma) and Clostron (e.g. WO 2007/148091), and integration of new pathway genes
through the use of 'allele coupled exchange' (ACE, e.g. WO 2009/101400); but deletion
of sections of DNA by such methods is laborious and these methods are not applicable
to the complete removal of endogenous plasmids.
Allelic exchange methods have been designed in order to generate clostridial strains
carrying specific point mutations or in-frame deletions through homologous
recombination methods. Due to the difficulties in isolating and selecting for successful
recombinant strains, these are generally inefficient, although they can be highly specific
(e.g. Cartman et al., Appl. Environ. Microbiol. 78(13), 4683-90 (2012)). Additionally RNA
knock-down and interference methods can be useful, as is transposon mutagenesis for
generating recombinant strains. However, although these are valuable research tools,
these methods target very specific or a narrow range of genes.
In some cases, the deletion of large, regionally targeted but non-specific sections of
genomic material is preferred as these methods can be used to generate strains in
which complete operons are removed or which can be used to identify essential versus
non-essential genetic material. In addition, in some circumstances, it is useful to be able
to quickly and efficiently remove endogenous plasmids. Removal of an endogenous
plasmid can answer questions regarding essentiality of the plasmid, e.g. a megaplasmid
may be essential for cell survival whereas a mini-plasmid may be dispensable. The
ability to quickly remove an endogenous plasmid can also be useful when origin
incompatibilities interfere with efficient transformation methods thereby making a
plasmid-free strain essential for research and exploitation. The loss of these native
plasmids 'naturally' may take considerable effort through multiple rounds of passaging
and selection.
Genome analysis of resultant plasmid-free or 'deletion carrying' strains may then be
used to understand more about microbial physiology which can be potentially linked to
substrate uptake mechanisms and production of solvents. For example C.
acetobutylicum carries a megaplasmid which encodes both an a-amylase enzyme and
the sol operon which is essential for solvent production. In some circumstances this
megaplasmid can be lost through strain degeneration. In C. acetobutylicum loss of the
megaplasmid provides a starting strain (M5) for manipulating metabolic flux. This can be
used to generate strains with altered ratios of acetone: butanol: ethanol solvents, by
complementing back only selected genes of the sol operon that were removed with the
loss of the plasmid (e.g. Nair et al., J. Bacterid., 176(18), 5843-6, 1994; Sillers et al.,
etab. Eng., 10(6), 321-32, 2008; Lee et al., Biotechnol. J., 4(10), 1432-1440, 2009).
A method which could potentially be used make these genome changes is
Transcriptional Activator-Like Endonucleases (TALENs, e.g. US 8,420,782 B2) but the
technology has been developed for editing eukaryotic genomes and has not yet been
specifically adapted for use in industrially-relevant solventogenic clostridial strains. The
need to engineer TALENs for each gene target is costly and time-consuming, and the
practicalities of precisely how the technology will work in Clostridia all count against it
becoming a widely accessible tool in the near future.
Therefore in summary the presently available methods are all complicated, multi-stage
and time-intensive procedures for generating highly specific mutations and deletions. In
order to enable more rapid screening or investigation of the importance of a variety of
chosen genes, operons, and plasmids, a method for simply and rapidly removing
fragments of genetic material from within bacterial genomic DNA or from plasmids
endogenously present in bacteria in a manner that does not need to be precise would
be much more useful.
A novel method has therefore been developed which is based on using the clostridial
CRISPR/Cas system. (CRISPR is an acronym for Clustered, Regularly Interspaced,
Short, Palindromic Repeats.) These systems are usually described as 'prokaryotic
adaptive immune systems' and are the means by which a bacterial or archaeal cell can
protect itself from invading DNA, usually phage or plasmid DNA.
Cells with a CRISPR/Cas system are able to selectively integrate short fragments from
'invading' DNA into the Cas gene cluster. Each fragment is called a 'Spacer' and is
flanked by direct repeats. If the cell encounters the same invading DNA again, it will
recognise it as hostile and will destroy it by cleaving it with the Cas endonuclease.
The sequence that the CRISPR/Cas system recognises in the invading DNA is called
the 'Protospacer' and has identity to the Spacer copy in the genome. In order to make
sure that the cell does not accidently attack the genomic copy of the Spacer, the
Protospacer in Cas I or Cas I I systems must have a short sequence associated with it
called the PAM sequence. The PAM sequence may be up- or down-stream of the
Protospacer sequence depending on the type of system. If it is not present or is mutated
in any way, the invading DNA will no longer be recognised by the cell and it will not be
destroyed.
The PAM sequence associated with cas9 from Streptococcus pyogenes is well known
(Jiang ef al., Nature Biotech. (March 2013), vol. 3 1, no. 3, pp. 233-239); however, the
PAM associated with clostridial systems has not previously been identified.
Not all prokaryotes have CRISPR/Cas gene homologues and of those that do they fall
into several distinct classes (Makarova et al., Nat. Rev. Microbiol., 9(6), 467-77. 201 ).
A lot of work has been published on the Type I I cas 9 system from Streptococcus
pyogenes and Streptococcus pneumoniae (e.g. Jiang et al., Nature Biotech. (March
2013), vol. 3 1, no. 3, pp. 233-239). This has been developed into a genome-editing tool
for use in eukaryotic cells, which has been used successfully in e.g. yeast (DiCarlo et al.,
Nucleic Acids Research, 41(7), 4336-4343, 2013), zebrafish (Hwang et al., Nat.
Biotechnol., 31(3), 227-9. 2013) and mammalian cells (Ran et al., Nature Protocols, 8,
2281-2308, 2013).
In eukaryotes, dsDNA breaks generated by CRISPR/Cas systems can be used to
generate deletions and other INDELs (INSertions and DELetions in DNA) to effectively
knock out virtually any gene, because of the presence of nonhomologous end joining
(NHEJ) pathways in the eukaryote cells. However, most bacteria lack these no n
homologous end joining (NHEJ) repair mechanisms, with Cas9 cleavage of genomic
sequences believed to lead only to cell death, rather than generation of gene deletions.
This was summarised in a review by Barrangou and Marraffini, Molecular Cell, 54(2),
234-244, 2014.
A new process has now been developed which enables the production of deletions in
bacterial genomes in a quick and efficient single-step process.
This new process can also be used to remove target endogenous plasmids (including
megaplasmids) from bacteria. By targeting two or more locations within the plasmid
sequence with the Deletion Vector, the system should cleave the plasmid into two or
more linear fragments which should be susceptible to destruction by native cell
mechanisms. Given the relatively small size of a plasmid compared to a typical genome,
complete degradation of the latter is more likely to occur before rejoing can take place,
resulting in loss of the plasmid This could be used to determine if endogenous
(mega)plasmids are essential for host survival, and may provide insight into the benefits
or metabolic burden of the plasmid to the host cell. In practice, the minimum number of
cut sites required to eliminate an endogenous plasmid is likely to be dependent on the
size of the target plasmid, as all of the resulting fragments should be sufficiently small
that they are degraded rather than rejoined.
In one embodiment, therefore, the invention provides a process for producing a deletion
in a bacterial genome, wherein the bacteria comprise a CRISPR/Cas system,
the process comprising the steps:
(a) transforming a population of bacteria with one or more Deletion Vectors, wherein the
Deletion Vector(s) are capable of directing production of first and second crRNAs which
target first and second PAM/Protospacers within the genomes of the bacteria within the
population;
(b) culturing the population of bacteria under conditions whereby the first and second
crRNAs are produced, wherein they target the first and second PAM/Protospacers, and
wherein the first and second crRNAs promote a dual cleavage of the genome in one or
more bacteria within the population, and wherein the two cleaved ends of the bacterial
genomic DNA rejoin; and
(c) isolating one or more bacteria whose genomes comprise a deletion in the bacterial
genomic DNA between the first and second PAM/Protospacers.
In step (c), the deletion in the bacterial genomic DNA preferably comprises at least the
region including and between the first and second PAM/Protospacers.
In some embodiments, one Deletion Vector is capable of directing production of both
the first and second crRNAs which target first and second PAM/Protospacers within the
genomes of the bacteria within the population.
Preferably the Deletion Vector comprises first and second Cas Spacer Elements,
flanked by Cas Direct Repeat Elements.
The first and second CRISPR Spacers may be in a single Cas array or in separate Cas
arrays within the Deletion Vectors.
In this embodiment, the Deletion Vector preferably comprises:
(i) a Cas Leader Element,
(ii) a first Cas Direct Repeat Element,
(iii) a first Cas Spacer Element which is capable of directing production of the
first crRNA,
(iv) a second Cas Direct Repeat Element,
(v) a second Cas Spacer Element which is capable of directing production of the
second crRNA, and
(vi) a third Cas Direct Repeat Element.
Alternatively, the Deletion Vector preferably comprises two arrays, the first array
comprising:
(i) a first Cas Leader Element,
(ii) a first Cas Direct Repeat Element,
(iii) a first Cas Spacer Element which is capable of directing production of the
first crRNA, and
(iv) a second Cas Direct Repeat Element,
and the second array comprising:
(v) a second Cas Leader Sequence,
(vi) a third Cas Direct Repeat Element,
(vii) a second Cas Spacer Element which is capable of directing production of the
second crRNA, and
(viii) a fourth Cas Direct Repeat Element.
In other embodiments, a first Deletion Vector is capable of directing production of the
first crRNA which targets the first PAM/Protospacers within the genomes of the bacteria
within the population; and a second Deletion Vector is capable of directing production of
the second crRNA which targets the second PAM/Protospacers within the genomes of
the bacteria within the population.
In this embodiment, the first Deletion Vector preferably comprises:
(i) a first Cas Leader Element,
(ii) a first Cas Direct Repeat Element,
(iii) a first Cas Spacer Element which is capable of directing production of the
first crRNA,
(iv) a second Cas Direct Repeat Element,
and/or the Second Deletion Vector preferably comprises:
(v) a second Cas Leader Element,
(vi) a third Cas Direct Repeat Element,
(vii) a second Cas Spacer Element which is capable of directing production of
the second crRNA,
(viii) a fourth Cas Direct Repeat Element.
In a further embodiment, the invention provides a process for producing a
rearrangement in a bacterial genome, wherein the bacteria comprise a CRISPR/Cas
system, the process comprising the steps:
(a) transforming a population of bacteria with one or more Deletion Vectors, wherein the
Deletion Vector(s) are capable of directing production of first and second crRNAs which
target first and second PAM/Protospacers within the genomes of the bacteria within the
population;
(b) culturing the population of bacteria under conditions whereby the first and second
crRNAs are produced, wherein they target the first and second PAM/Protospacers, and
wherein the first and second crRNAs promote a dual cleavage of the genome in one or
more bacteria within the population; and
(c) isolating one or more bacteria whose genomes comprise a rearrangement in the
bacterial genomic DNA compared to control non-transformed bacteria.
In another embodiment, the invention provides a process for removing or deleting DNA
from an endogenous plasmid in a bacterium (which is likely to result in a loss of the
plasmid), wherein the bacteria comprise a CRISPR/Cas system, the process comprising
the steps:
(a) transforming a population of bacteria with one or more Deletion Vectors,
wherein the Deletion Vector(s) are capable of directing production of two or more
crRNAs which target two or more PAM/Protospacers within the target plasmids in the
bacteria within the population;
(b) culturing the population of bacteria under conditions whereby the two or more
crRNAs are produced and wherein the two or more crRNAs target the one or more
PAM/Protospacers, and wherein the two or more crRNAs promote the cleavage of the
target plasmids at two or more locations to produce linearised DNA fragments in two or
more bacteria within the population, and wherein the linearised fragments are subject to
degradation by endogenous cell mechanisms; and
(c) isolating one or more bacteria who lack the target plasmid.
Preferably, the linearised fragments are subject to degradation by endogenous cell
mechanisms before the (inefficient) end rejoining processes are able to repair the
plasmid.
The bacteria in the population of bacteria must have a CRISPR/Cas system. This
CRISPR/Cas system will be one which is capable of cleaving the chromosomal DNA or
other target DNA, e.g. target plasmid, preferably endogenous plasmid of the bacteria
using the first and second crRNAs or the two or more crRNAs.
It will be accepted that there may, in some cases, be contamination within bacterial
populations. As used herein, the term "population of bacteria" refers primarily to the
bacteria which it is desired to transform with the Deletion Vector.
Preferably, the CRISPR/Cas system is a Type I CRISPR/Cas system.
The bacteria in the population may have an endogenous CRISPR/Cas system or the
CRISPR/Cas system may be heterologous. For example, a heterologous CRISPR/Cas
system may be plasmid-based.
Preferably, the CRISPR/Cas system is an endogenous CRISPR/Cas system, i.e. it is
present in the wild-type bacteria. In some embodiments of the invention, the
CRISPR/Cas system is not a plasmid-based system.
The bacteria in the population may, for example, be Gram-positive or Gram-negative
bacteria. Preferably the bacteria are Gram-positive.
In some embodiments, the bacteria are spore-forming bacteria. In other embodiments,
the bacteria are saccharolytic bacteria.
The bacteria may be aerobic or anaerobic bacteria. Preferably, the bacteria are
anaerobic bacteria. The bacteria may be thermophilic bacteria.
In yet other embodiments, the bacteria are able to convert a substrate into RCOOH, for
example, into acetate and/or butyrate. In this context, R is an aliphatic C1-C5,
preferably C1-3, alkyl or alkenyl group. The bacteria may also be able to convert the
RCOOH into a solvent, preferably into one or more of acetone, ethanol and/or butanol.
In other embodiments, the bacteria are solvent-producing bacteria. As used herein, the
term "solvent-producing" means that the bacteria are those which are capable of
producing a solvent, preferably a solvent such as acetone, ethanol, propanol and/or
butanol. In certain particularly preferred embodiments, the bacteria are capable of
producing ethanol, acetone and butanol. Preferably, the bacteria are butanol-producing
bacteria or butanol-tolerant bacteria.
In some preferred embodiments, the bacteria are of the genus Clostridium. Preferred
Clostridium species include C. acetobutylicum, C. arbusti, C. aurantibutyricum, C.
beijerinckii, C. cellulovorans, C. cellulolyticum, C. thermocellum, C. thermobutyricum, C.
pasteurianum, C. kluyveri, C. novyi, C. saccharobutylicum, C. thermosuccinogenes, C.
thermopalmarium, C. saccharolyticum, C. saccharoperbutylacetonicum, C.
tyrobutyricum, C. tetanomorphum, C. magnum, C. Ijungdahlii, C. autoethanogenum, C.
butyricum, C. puniceum, C. diolis, C. homopropionicum and C. roseum.
In some preferred embodiments of the invention, the bacteria are C.
saccharoperbutylacetonicum strain N 1, e.g. N1-4. In other embodiments of the invention,
the bacteria are C. saccharoperbutylacetonicum N1-4 (HMT). In yet other embodiments
of the invention, the host cell is C. saccharoperbutylacetonicum N1-504.
In other preferred embodiments, the bacteria are C. pasteurianum (e.g. DS 525), C.
tyrobutyricum (e.g. ATCC 52755), or C. saccharobutylicum (e.g. NCP 258 and NCP 262)
or Clostridium sp. DL-VIII.
In other preferred embodiments, the bacteria are of the genus Bacillus. In other
preferred embodiments, the bacteria are of the order Actinomycetales. In other
embodiments, the bacteria are preferably not Streptococcus or E. coli.
The bacteria are preferably ones which have an endogenous end-joining enzyme or
mechanism. In other embodiments, the bacteria are ones which have an heterologous
end-joining enzyme or pathway.
The target plasmid may be an endogenous plasmid (which occurs naturally in the
bacteria in question).
The core region of bacterial DNA (genomic or plasmid) to be deleted is flanked by two
CRISPR PAM/Protospacers which are capable of being recognised by the bacteria's
CRISPR/Cas system.
A PAM/Protospacer is a sequence in the bacterial genome or target plasmid that
includes a functional combination of a PAM sequence and a Protospacer. Each
PAM/Protospacer sequence is capable of being recognised by the CRISPR/Cas system
that is being used and, upon production of the appropriate crRNA, it will be cleaved by
the CRISPR/Cas system.
As used herein, the term "functional CRISPR/PAM Protospacer" means a CRISPR
PAM/Protospacer which is capable of being recognised by a crRNA which recognises a
CRISPR/PAM Protospacer in the bacterial genome or target plasmid. In some cases, a
single mutation (e.g. in the PAM sequence) may be enough to render the CRISPR/PAM
Protospacer non-functional.
PAM is an abbreviation for Protospacer-Adjacent Motif. PAM Elements are capable of
being recognised by the bacterial CRISPR/Cas system. PAM Elements are generally 3-
6 nucleotides long and are specific to each bacterial species.
The orientation of the PAM Element with respect to the Protospacer in the bacterial
genome or target plasmid is important. In some bacterial species, the PAM Element is
generally found at or near the 5' end of the Protospacer; in other species, the PAM
Element is generally found at or near the 3' end of the Protospacer.
The PAM Element may be on either strand of the bacterial genome or target plasmid
but the sequence chosen as the Cas Spacer Element should be on the same DNA
strand as the PAM Element (so that the PAM Element and Protospacer are directly
adjacent).
Some studies have found that almost any mutation in the PAM Element eliminates
recognition by the CRISPR/Cas system (e.g. Jiang et al., Nature Biotech (March 201 3),
vol. 3 1, no. 3, pp. 233-239). The PAM/Protospacers must each have a functional PAM
Element, in addition to a functional Protospacer. As used herein, the term "functional
PAM Element" or "CRISPR PAM Element which is functional in the bacteria" means that
the PAM Element is capable of being recognised by the bacteria's endogenous
CRISPR/Cas system or, if the bacteria do not have an endogenous CRISPR/Cas
system, by the vector-based heterologous CRISPR/Cas system which has been
introduced into the bacteria.
More than one sequence might be able to function as the PAM Element in the chosen
bacterial species. For example, the l-E CRISPR-Cas system from Escherichia
coli K-12 is known to have four functional PAM sequences (Gomaa et al. (2014). mBio,
5 ( 1 ) : e00928-13 DOI: 10.1 128/mBio.00928-13), and in C. saccharoperbutylacetoniucm
N1-4 (HMT), at least four effective PAM sequences (CCC, CCT, CCA and CCG) have
been identified using the method described in Example 2.
The ability of a PAM Element to function in a particular bacterial species may be tested
by transforming the bacteria having a CRISPR/Cas system (either its endogenous
CRISPR/Cas system or a heterologous plasmid-derived system) with a plasmid
comprising a CRISPR Spacer, and an adjacent test-PAM Element. If the PAM Element
is functional in the bacteria, the PAM Element-containing plasmid will be destroyed by
the CRISPR/Cas system and the transformation efficiency will be significantly reduced.
The concept is illustrated herein in Example 2.
The CRISPR Protospacers are the sequences within the bacterial genome or target
plasmid which are targeted by the crRNAs (provided that compatible PAM Elements are
also appropriately located).
In step (a) of the process for producing a deletion in a bacterial genome, a population of
bacteria are transformed with one or more Deletion Vectors, wherein the Deletion
Vector(s) are capable of directing production of first and second crRNAs which target
first and second PAM/Protospacers within the genomes of the bacteria within the
population.
The aim of this step (a) is to prepare for the production two crRNAs: the first crRNA will
bind to the first PAM/Protospacer in the bacterial genome; and the second crRNA will
bind to the second PAM/Protospacer in the bacterial genome.
In step (a) of the process for removing a target plasmid in a bacterium, a population of
bacteria are transformed with one or more Deletion Vectors, wherein the Deletion
Vector(s) are capable of directing production of two or more crRNAs which target two or
more PAM/Protospacers within the target plasmids in the bacteria within the population.
The aim of this step (a) is to prepare for the production the two or more crRNAs which
will bind to the one or more PAM/Protospacers in the target plasmid.
ln the process for removing a target (preferably endogenous) plasmid in a bacterium,
the Deletion Vector may be capable of directing production of one, two, three, four, five
or more crRNAs. These may target one, two, three, four, five or more
PAM/Protospacers within the target plasmids.
As used herein, the term "transformation" and "transforming" refers to any step by which
the Deletion Vector(s) are inserted into the bacterial cells. Hence it includes any form of
electroporation, conjugation or transfection, inter alia.
The deletion may be in-frame or not in-frame. The PAM/Protospacers will both / all be
within the site of the deletion. Preferably, the deletion includes at least part of the PAM
or PAM/Protospacer so that the PAM/Protospacer becomes no longer functional (i.e. no
longer recognised by the CRISPR/Cas system).
The Deletion Vector is preferably a circular vector.
The Deletion Vector preferably has an Origin Element, most preferably a Gram positive
Origin Element (for example "pBP1").
The Deletion Vector may also comprise an appropriate selection marker (e.g. antibiotic
resistance gene).
The Deletion Vector does not comprise a CRISPR PAM/Protospacer which is capable
of being recognised by a crRNA which recognises the CRISPR/PAM Protospacer in the
bacterial genome (or target plasmid).
In the process for producing a deletion in a bacterial genome, step (b) comprises
culturing the population of bacteria under conditions whereby the first and second
crRNAs are produced, wherein they target the first and second PAM/Protospacers and
wherein the first and second crRNAs promote the cleavage of the genome in one or
more bacteria within the population, and wherein the two cleaved ends of the bacterial
genomic DNA rejoin.
In this step, either both of the CRISPR PAM/Protospacers are removed from the
bacterial genome (in the deletion) or both of the CRISPR PAM/Protospacers are
rendered incapable of being recognised by crRNAs which recognise the
PAM/Protospacers.
In this step, the bacterium's CRISPR/Cas system (either endogenous or heterologous)
will cleave the bacterial genomic DNA at a site within the genomic sequence that
corresponds to the first PAM/Protospacer and at a site within the genomic sequence
that corresponds to the second PAM/Protospacer. Hence the bacterial genome will be
cut twice, linearised and a deleted section of DNA will be liberated. The actual sites of
cleavage within the first and second PAM/Protospacers, and hence the extent of the
deletion, will be dependent on the distance between the two PAM/Protospacers in the
genome. It is very possible that the cleaved ends of the genomic DNA will be trimmed
back by cellular mechanisms, prior to rejoining. In some cases, the extent of this
trimming back may be quite significant, for example resulting in loss of many kb of
sequence flanking the pair of PAM/Protospacer. The maximum extent of trimming could
be determined by the proximity of essential genes, as trimming back further (into such
genes) would not be viable, and will be in competition with process(es) for end rejoining.
In the process for removing a target plasmid in a bacterium, step (b) comprises
culturing the population of bacteria under conditions whereby the two or more crRNAs
are produced and wherein the two or more crRNAs target the two or more
PAM/Protospacers, and wherein the two or more crRNAs promote the cleavage of the
target plasmids at two or more locations to produce linearised DNA fragments in one or
more bacteria within the population, and wherein the linearised fragments are subject to
degradation by endogenous cell mechanisms. In cases where the degradation occurs
more quickly than the DNA rejoining, the plasmid will be lost from the cell.
ln this step, the target plasmid is cleaved at or near the CRISPR PAM/Protospacers to
produce two or more linear fragments of the plasmid. Such fragments may be degraded
by host cell mechanisms before the host repair mechanisms are able to rejoin the DNA.
Suitable conditions for culturing the bacteria will be readily known in the art.
Such conditions are, for example, described in "Clostridia: Biotechnology and Medical
Applications", Eds H. Bahl and P. Durre, ISBN 3-527-30175-5, especially section 3.4
"Growth conditions and nutritional requirements". Details are also given in Bergey's
Manual of Systematic Bacteriology, Volume appropriate to the chosen phylum of
bacteria, e.g. Volume Three for the Firmicutes, ISBN 978-0-387-95041-9.
The term "crRNA" means CRISPR RNA. crRNAs are short single-stranded RNA
molecules consisting of short Direct Repeat sequences flanking a target Spacer
sequence to be cleaved by the CRISPR/Cas system.
The "Cas Leader Element" is a DNA element which is generally found upstream of the
first repeat in the Direct Repeat cluster. It helps to promote the production of crRNA, i.e.
it functions as a RNA promoter. Numerous Cas Leader sequences have been identified
to date and its sequence may readily be established in any particular Cas system.
Preferably, the Cas Leader sequence is one which corresponds to the CRISPR/Cas
system which is present in the bacterial population which is being transformed.
The Cas Direct Repeat sequences are DNA elements which are recognised by the
CRISPR/Cas system which is present in the population of bacteria. These Direct
Repeats are generally 25-35 nucleotides in length, more generally about 29 or 30
nucleotides in length.
The Direct Repeats do not need to be of identical sequence (generally a difference of 1-
2 nucleotides is tolerated by the Cas protein). The Direct Repeats generally have
palindromic regions which are capable of forming hair-pin loops.
The DNA sequence of Direct Repeats which are suitable for any one CRISPR/Cas
system may readily be found from any inspection of the CRISPR/Cas direct repeat-
Spacer cluster of that system.
The Cas Spacer Element comprises a sequence of 20-50 nucleotides (preferably 30-40,
more preferably 36-38 nucleotides) with a high level of sequence identify to the 20-50
nucleotides (preferably 30-40, more preferably 36-38 nucleotides) which are found
(preferably immediately) 5' to the PAM Element in the PAM/Protospacer or (preferably
immediately) 3' to the PAM Element in the PAM/Protospacer, depending on the
preference of the CRISPR/Cas system which is present in the bacterial population of
interest.
Preferably, the PAM Elements in the bacterial genome or target plasmids are directly
adjacent to the start of the Protospacer sequences (in the bacterial genome).
The degree of sequence identity between the Protospacers in the genomic DNA or
target plasmid and the Spacer Element sequences in the Deletion Vector(s) is each
preferably at least 80%, more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or
99%, or is 100%.
Preferably, the Cas Spacer sequence is selected such that there is a low probability of
interaction with a non-Protospacer Element.
The first and second Cas Spacer Elements bind to the first and second
PAM/Protospacers in the bacterial genome.
Similarly, the Cas Spacer Elements bind to corresponding sites in the target plasmid.
The minimum extent of the deletion is defined by the distance between the first and
second PAM/Protospacers.
Preferably, this distance is 10 bp-1000 b, more preferably 100 bp-100 kb, even more
preferably 200-500 bp.
The Deletion Vectors may comprise antibiotic-resistance elements or other selection
markers, thus allowing the Vectors to be selected for, e.g. in the presence of certain
antibiotics, for example chloramphenicol, erythromycin, tetracycline, spectinomycin,
streptomycin etc. Preferably, the Deletion Vectors (when more than one are present)
comprise antibiotic-resistance elements which allow for their selection on different
antibiotics.
Percentage amino acid sequence identities and nucleotide sequence identities may be
obtained using the BLAST methods of alignment (Altschul ei al. (1997), "Gapped
BLAST and PSI-BLAST: a new generation of protein database search programs",
Nucleic Acids Res. 25:3389-3402; and http://www.ncbi.nlm.nih.gov/BLAST). Preferably
the standard or default alignment parameters are used.
Standard protein-protein BLAST (blastp) may be used for finding similar sequences in
protein databases. Like other BLAST programs, blastp is designed to find local regions
of similarity. When sequence similarity spans the whole sequence, blastp will also report
a global alignment, which is the preferred result for protein identification purposes.
Preferably the standard or default alignment parameters are used. In some instances,
the "low complexity filter" may be taken off.
BLAST protein searches may also be performed with the BLASTX program, score=50,
wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST
(in BLAST 2.0) can be utilized as described in Altschul e al. ( 1997) Nucleic Acids Res.
25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated
search that detects distant relationships between molecules. (See Altschul ei al. (1997)
supra). When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of
the respective programs may be used.
With regard to nucleotide sequence comparisons, MEGABLAST, discontiguous
megablast, and blastn may be used to accomplish this goal. Preferably the standard or
default alignment parameters are used. MEGABLAST is specifically designed to
efficiently find long alignments between very similar sequences. Discontiguous
MEGABLAST may be used to find nucleotide sequences which are similar, but not
identical, to the nucleic acids of the invention.
The BLAST nucleotide algorithm finds similar sequences by breaking the query into
short subsequences called words. The program identifies the exact matches to the
query words first (word hits). The BLAST program then extends these word hits in
multiple steps to generate the final gapped alignments. In some embodiments, the
BLAST nucleotide searches can be performed with the BLASTN program, score=100,
wordlength=12.
One of the important parameters governing the sensitivity of BLAST searches is the
word size. The most important reason that blastn is more sensitive than MEGABLAST is
that it uses a shorter default word size ( 1 ) . Because of this, blastn is better than
MEGABLAST at finding alignments to related nucleotide sequences from other
organisms. The word size is adjustable in blastn and can be reduced from the default
value to a minimum of 7 to increase search sensitivity.
A more sensitive search can be achieved by using the newly-introduced discontiguous
megablast page (www.ncbi.nlm.nih.gov/Web/Newsltr/FallWinter02/blastlab.html). This
page uses an algorithm which is similar to that reported by Ma et al. (Bioinformatics.
2002 Mar; 18(3): 440-5). Rather than requiring exact word matches as seeds for
alignment extension, discontiguous megablast uses non-contiguous word within a
longer window of template. In coding mode, the third base wobbling is taken into
consideration by focusing on finding matches at the first and second codon positions
while ignoring the mismatches in the third position. Searching in discontiguous
MEGABLAST using the same word size is more sensitive and efficient than standard
blastn using the same word size. Parameters unique for discontiguous megablast are:
word size: 1 or 12; template: 16, 18, or 2 1; template type: coding (0), non-coding (1),
or both (2).
The two cleaved ends of the bacterial genome will be rejoined by bacterial DNA repair
mechanisms. (The skilled artisan will readily appreciate that the references herein to
"two cleaved ends" refer to the two double-stranded DNA ends of the bacterial genome,
which technically comprise four DNA strands.)
Smaller fragments, for example generated from cleavage of megaplasmids or plasmids
may not rejoin, but instead be degraded by cellular mechanisms before rejoining can
occur.
The deletion will often be a crude deletion, i.e. the deletion will often not be a precise
rejoining between the two ends of the bacterial genome which have been cut by the Cas
enzyme. For example, the cell repair mechanism may process the cut ends resulting in
the deletion of one or potentially many more additional nucleotides. For example, the
deletion may comprise a deletion of 1-5 kb, 5-10 kb, 10-30 kb, 30-50 kb or 50-1 00 kb
DNA on one or both sides of the region of DNA flanked by the first and second
PAM/Protospacer sites.
In the process for producing a deletion in a bacterial genome, step (c) comprises
isolating one or more bacteria whose genomes comprise a deletion in the bacterial
genomic DNA between and optionally including the first and second PAM/Protospacers.
Preferably, the deletion comprises the first and second PAM/Protospacers in addition to
at least 1-10 kb of flanking DNA on each side of the region of DNA flanked by the first
and second PAM/Protospacer sites.
Bacteria having the desired DNA deletion will easily lose the deleted DNA (between and
optionally including the two CRISPR/Cas cut sites) due to the fact that it will be linear
DNA and thus prone to degradation by host cell mechanisms (e.g. digested by
endogenous nucleases). It is likely that the deleted section of DNA will not contain all of
the essential genetic elements for stable maintenance in the cell (e.g. appropriate origin)
even if it were to be re-joined before degradation.
The isolated bacteria are ones wherein the two cleaved ends of the genome have been
rejoined.
In the process for removing an endogenous plasmid, step (c) comprises isolating one or
more bacteria that lack the intact or uncleaved target plasmid that was previously
carrying the one or more PAM/Protospacers.
The bacteria which are selected for or isolated will be live bacteria.
The invention further provides a process for making mutated bacteria, which comprises
producing a deletion in a bacterial genome by a process of the invention.
In addition, the invention further provides a process for making mutated bacteria which
lack target megaplasmids or other plasmids, preferably an endogenous plasmid, by a
process of the invention.
In plasmids, it is possible that the method will generate a deletion that does not lead to
elimination of the complete plasmid, but instead results in generation of a smaller
plasmid lacking a section of DNA including the two (or more) target sites. This is more
likely to be seen with larger plasmids, since they are more likely to be able to tolerate a
large deletion than a smaller plasmid, as there is less chance of the deletion affecting
essential elements, such as a suitable origin.
The invention also provides bacteria whose genomes have had a deletion which was
produced by a process of the invention.
In addition, the invention also provides bacteria who have been caused to lose the
target megaplasmid or plasmid, preferably an endogenous (mega)plasmid, by a process
of the invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows an alignment of Direct Repeat sequences from a number of clostridial
species.
Figure 2 shows the effect of the PAM sequence on the transformation efficiency of
plasmids into C. saccharoperbutylacetonicum N1-4 (HMT).
Figure 3 compares the wild-type sequence of C. saccharoperbutylacetonicum N1-4
(HMT) with the same region of genomic sequence data from one of the deletion strains
produced in Example 3. The diagram was prepared using Artemis genome browsing
tools at: https://www.sanger.ac.uk/resources/software/artemis/.
EXAMPLES
Example : Alignment of Direct Repeat sequences from a number of clostridial
species
Aim : To identify some Direct Repeat sequences that could be used in the process of the
invention.
Method:
Direct Repeats and the Spacer sequences were found using the CRISPRFinder
programme Grissa, I., Vergnaud, G., & Pourcel, C. (2007). CRISPRFinder: a web tool to
identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res.,
35, W52-7.
Results:
A selection of Direct Repeat sequences from a number of clostridial species are
displayed in Figure . In some cases the specific strain has more than one sequence so
the most frequently used Direct Repeat sequence(s) is included here. Abbreviations are
as follows: C_saccharoper = Clostridium saccharoperbutylacetonicum N1-4 (HMT) or
N1-504), C_saccharob = Clostridium saccharobutylicum (NCP258 or NCP262, _ 1 and
2 refer to the 2 main DR clusters), C yro = Clostridium tyrobutyricum (ATCC 52755,
_ 1 and 2 refer to the 2 main DR clusters), C_pasteurianum = Clostridium pasteurianum
(DSM 525), C_autoethanogenum = Clostridium autoethanogenum (DSM10061 ) ,
C_sp_DLVI 11= Clostridium sp. (DL-VI 11) .
Example 2 : Confirming the PAM sequence in C. saccharoperbutylacetonicum
N1-4 (HMT)
Aim : To demonstrate how to test effectiveness of putative PAM sequences.
Method :
The sequence of Spacer_53 from the main Direct Repeat cluster of C.
saccharoperbutylacetonicum N1-4 (HMT) was cloned into the clostridial shuttle vector,
pMTL83251 . Immediately adjacent to the 5' end of this Spacer Element various different
trinucleotide combinations were incorporated, including the predicted PAM sequences
CCC, CCG, CCT and a non-PAM sequence GAC. When correctly combined with a
functional PAM sequence, the Spacer Element functions as a Protospacer.
The plasmids were transformed into C. saccharoperbutylacetonicum N1-4 (HMT) using
standard electroporation protocols followed by an overnight recovery stage in Clostridial
Growth Medium (CGM) also containing 5% glucose. The mixture was then spread onto
CGM agar plates containing 5% glucose and 40 g/ml erythromycin and left for at least
48 hours in an anaerobic cabinet at 32°C. Colonies were then counted to determine the
change in transformation efficiency compared with transformation of the empty vector.
CGM medium was prepared by dissolving the following amounts in 750 ml dH20 : 5.0 g
yeast extract, 0.75 g K2HP0 4, 0.75 g KH2P0 4 > 0.40 g MgS0 4.7H20 , 0.01 g FeS0 4.7H20 ,
0.01 g MnS0 4.4H20 , 1.0 g NaCI, 2.0 g (NH4)2S0 , 2.0 g asparagine (and 15 g
bacteriological agar no.1 if making solid medium) and autoclaved. The pH of the
medium was not adjusted (usually in the region of 6.6). A 20 % (w/v) glucose solution
(50 g glucose dissolved in sufficient dH20 to give a final volume of 250 ml) was
prepared and autoclaved separately. Once cool, the glucose and CGM solutions were
combined as needed.
Results :
The relative efficiencies of transformation of the different plasmids are presented in
Figure 2. Both the empty plasmid pMTL83251 and the plasmid carrying Spacer_53
without a PAM sequence gave a lawn of colonies. Plasmids carrying Spacer_53
adjacent to a 5' CCC (PAMC), CCT (PAMT) or CCA (PAMA) yielded significantly fewer
colonies.
Example 3 : Demonstrating the crude deletion method
Aim : To show how the process of the invention can be adapted to delete a large
fragment of DNA.
Method:
A selection of candidate Protospacer Elements (located immediately 3' of PAM
sequences known to be functional in this bacterial system) were identified in a single
target gene, and from these two were chosen that were approximately 1 kb apart (in the
genomic DNA), named, for this Example, as ProtospaceM and Protospacer_2, and
being located 5' and 3' in the target gene, respectively. A Deletion Vector was designed
and constructed based on a pMTL82154 backbone. It carried the leader sequence, the
first Direct Repeat, Spacer_1 (corresponding to potential ProtospaceM), a second
Direct Repeat, Spacer_2 (corresponding to potential Protospacer_2) and a third Direct
Repeat.
This Deletion Vector was transformed into C. saccharoperbutylacetonicum N1-4 (HMT)
using standard electroporation methods. When transformed into the cells the Deletion
Vector resulted in the Cas-mediated cleavage of the gene and subsequent loss of
genetic material.
Two colonies were recovered using this method, one of which was subsequently
sequenced. This revealed that method of the invention had generated a 29205 nt
deletion, compared to the parental strain. The deleted section included all of the 1 kb
region between the two targeted PAM/Protospacer sites (see Figure 3).
In the genome sequence of the mutant strain, the sequence data reading across the
deletion site was as follows:
A ACCCIA TT CCA A
TTTATACAAAACTCTTC (SEQ I D NO : 12 )
The first 27 nucleotides, underlined with a wavy line, match the wildtype genome in
position 5709460 to 5709486. The last 74 nucleotides, underlined with a dashed line,
match the wildtype genome in position 5738692 to 5738765. The total length of the
deletion was 5738692-1-5709486 = 29205 nt.

CLAIMS
1. A process for producing a deletion in a bacterial genome, wherein the bacteria
comprise a CRISPR/Cas system, the process comprising the steps:
(a) transforming a population of bacteria with one or more Deletion Vectors, wherein the
Deletion Vector(s) are capable of directing production of first and second crRNAs which
target first and second PAM/Protospacers within the genomes of the bacteria within the
population;
(b) culturing the population of bacteria under conditions whereby the first and second
crRNAs are produced, wherein they target the first and second PAM/Protospacers, and
wherein the first and second crRNAs promote the dual cleavage of the genome in one
or more bacteria within the population, and wherein the two cleaved ends of the
bacterial genomic DNA rejoin; and
(c) isolating one or more bacteria whose genomes comprise a deletion in the bacterial
genomic DNA between the first and second PAM/Protospacers.
2. A process as claimed in claim 1, wherein one Deletion Vector is capable of
directing production of both the first and second crRNAs which target the first and
second PAM/Protospacers within the genomes of the bacteria within the population.
3. A process as claimed in claim 2, wherein the Deletion Vector comprises first and
second Cas Spacer Elements which are flanked by Cas Direct Repeat Elements, which
encode the first and second crRNAs.
4. A process as claimed in claim 3, wherein the first and second CRISPR Spacer
Elements are in a single Cas array within the Deletion Vector or in separate Cas arrays
within the Deletion Vector.
5. A process as claimed in claim 4, wherein the Deletion Vector comprises:
(i) a Cas Leader Element,
(ii) a first Cas Direct Repeat Element,
(iii) a first Cas Spacer Element which is capable of directing production of the
first crRNA,
(iv) a second Cas Direct Repeat Element,
(v) a second Cas Spacer Element which is capable of directing production of the
second crRNA, and
(vi) a third Cas Direct Repeat Element.
6. A process as claimed in claim 4, wherein the Deletion Vector comprises two
arrays, the first array comprising:
(i) a first Cas Leader Element,
(ii) a first Cas Direct Repeat Element,
(iii) a first Cas Spacer Element which is capable of directing production of the
first crRNA,
(iv) a second Cas Direct Repeat Element,
and the second array comprising:
(v) a second Cas Leader Element,
(vi) a third Cas Direct Repeat Element,
(vii) a second Cas Spacer Element which is capable of directing production of
the second crRNA, and
(viii) a fourth Cas Direct Repeat Element.
7. A process as claimed in claim 1, wherein a first Deletion Vector is capable of
directing production of the first crRNA which targets the first PAM/Protospacers within
the genomes of the bacteria within the population; and a second Deletion Vector is
capable of directing production of the second crRNA which targets the second
PAM/Protospacers within the genomes of the bacteria within the population.
8. A process as claimed in claim 7, wherein the first Deletion Vector comprises:
(i) a first Cas Leader Element,
(ii) a first Cas Direct Repeat Element,
(iii) a first Cas Spacer Element which is capable of directing production of the
first crRNA,
(iv) a second Cas Direct Repeat Element,
and/or the Second Deletion Vector comprises:
(v) a second Cas Leader Element,
(vi) a third Cas Direct Repeat Element,
(vii) a second Cas Spacer Element which is capable of directing production of
the second crRNA,
(viii) a fourth Cas Direct Repeat Element.
9. A process as claimed in any one of the preceding claims, wherein in step (b),
either both of the PAM/Protospacers are removed from the bacterial genome or both of
the PAM/Protospacers are rendered incapable of being recognised by crRNAs which
recognise the PAM/Protospacers.
10. A process for removing a target endogenous plasmid from bacteria, wherein the
bacteria comprise a CRISPR/Cas system, the process comprising the steps:
(a) transforming a population of bacteria with one or more Deletion Vectors,
wherein the Deletion Vector(s) are capable of directing production of two or more
crRNAs which target one or more PAM/Protospacers within the target plasmids in the
bacteria within the population;
(b) culturing the population of bacteria under conditions whereby the two or more
crRNAs are produced and wherein the two or more crRNAs target the two or more
PAM/Protospacers, and wherein the two or more crRNAs promote the cleavage of the
target plasmids at two or more locations to produce linearised DNA fragments in one or
more bacteria within the population, and wherein the linearised fragments are preferably
subject to degradation by endogenous cell mechanisms; and
(c) isolating one or more bacteria who lack the target plasmid.
11. A process as claimed in claim 10, wherein one, two, three, four or five crRNAs
target one, two, three, four or five PAM/Protospacers.
12. A process as claimed in claim 10 or claim 11, wherein the target plasmid is a
megap!asmid or an endogenous plasmid.
13. A process as claimed in any one of the preceding claims, wherein the bacteria
have an endogenous CRISPR/Cas system.
14. A process as claimed in any one of the preceding claims, wherein the bacteria
have a Type I CRISPR/Cas system.
15. A process as claimed in any one of the preceding claims, wherein the bacteria
are Gram positive bacteria.
16. A process as claimed in any one of the preceding claims, wherein the bacteria
are non-highly recombinogenic bacteria.
17. A process as claimed in any one of the preceding claims, wherein the bacteria
are of the class Clostridia preferably of the order Clostridiaceae, more preferably of the
genus Clostridium or where the bacteria are of the order Actinomycetales or of the
genus Bacillus.
18. A process as claimed in claim 17, wherein the bacteria is selected from the group
consisting of C. acetobutylicum, C. arbusti, C. aurantibutyricum, C. beijerinckii, C.
cellulovorans, C. cellulolyticum, C. thermocellum, C. thermobutyricum, C. pasteurianum,
C. kluyveri, C. novyi, C. saccharobutylicum, C. thermosuccinogenes, C.
thermopalmarium, C. saccharolyticum, C. saccharoperbutylacetonicum, C.
tyrobutyricum, C. tetanomorphum, C. magnum, C. ljungdahlii, C. autoethanogenum, C.
butyricum, C. puniceum, C. diolis, C. homopropionicum and C. roseum.
9. A process for making a mutated bacterium, which comprises producing a
deletion in a bacterial genome by a process as claimed in any one of claims 1 to 9 or 13
to 18.
20. A process for making a mutated bacterium, which comprises removing a target
endogenous plasmid in a bacterium by a process as claimed in any one of claims 10 to
18.
2 1. Bacteria whose genomes have had a deletion which was produced by a process
as claimed in any one of claims 1 to 9 or 13 to 18.
22. Bacteria from whom a target endogenous plasmid has been removed by a
process as claimed in any one of claims 10 to 18.