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.
1. A process for producing a deletion in a bacterial genome, wherein the bacterial genome is from a bacteria of the class Clostridia, and 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 bacteria are of the class Clostridia and wherein the bacteria comprise a CRISPR/Cas system, and 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 major fragment 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 Cas 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 claim 1 , 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 as claimed in claim 1 , wherein the bacteria have an endogenous CRISPR/Cas system.
11. A process as claimed in claim 1 , wherein the bacteria have a Type I CRISPR/Cas system.
12. A process as claimed in claim 1 , wherein the bacteria are of the genus Clostridium.
13. A process as claimed in claim 12 , 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.
14. A process for making a mutated bacterium, wherein the bacterium is of the class Clostridia, the process comprising producing a deletion in a bacterial genome, wherein the bacterial genome is from a bacteria of the class Clostridia, by a process as claimed in claim 1 .