Curing for recursive nucleic acid-guided cell editing
The present disclosure provides automated multi-module instrumentation and automated methods for performing recursive editing of live cells with curing of editing vectors from prior rounds of editing.
1. A composition of matter for recursive nucleic acid-directed nuclease CRISPR editing comprising one or more vectors, wherein the one or more vectors comprise:
(i) a DNA sequence encoding one or more editing gRNAs under the control of a first inducible promoter;
(ii) one or more donor DNA sequences, wherein the one or more editing gRNAs and the one or more donor DNA sequences edit one or more target sequences in a cell;
(iii) a curing target sequence, wherein the curing target sequence is capable of being cut to cleave the one or more vectors;
(iv) one or more selectable markers; and
(v) a DNA sequence encoding a curing gRNA sequence under the control of a second inducible promoter, wherein the curing gRNA sequence targets the curing target.
2. The composition of matter of claim 1 , wherein the first inducible promoter is a pL promoter.
3. The composition of matter of claim 1 , wherein the second inducible promoter is a pPhIF promoter.
4. The composition of matter of claim 1 , wherein the curing gRNA sequence comprises an anti-origin of replication gRNA sequence.
5. The composition of matter of claim 1 , wherein the curing target sequence comprises a pUC origin of replication.
6. The composition of matter of claim 1 , wherein the curing gRNA sequence comprises an anti-pUC origin of replication gRNA sequence.
7. The composition of matter of claim 1 , wherein the one or more selectable markers comprise an antibiotic resistant gene.
8. The composition of matter of claim 1 , wherein the one or more editing gRNAs and the one or more donor DNA sequences are designed to change one or more bases of a target genomic DNA sequence at one or more specific sites.
9. The composition of matter of claim 1 , wherein the one or more editing gRNAs and the one or more donor DNA sequences are designed to change 20 or more bases of a target genomic DNA sequence at one or more specific sites.
10. The composition of matter of claim 1 , wherein the one or more editing gRNAs and the one or more donor DNA sequences are designed to change at least 94 bases of a target genomic DNA sequence at one or more specific sites.
11. The composition of matter of claim 1 , wherein the one or more editing gRNAs and the one or more donor DNA sequences are designed to change 40 or more bases of a target genomic DNA sequence at one or more specific sites.
12. The composition of matter of claim 1 , wherein the one or more selectable markers are different.
13. The composition of matter of claim 1 , wherein the curing target sequence comprises the one or more donor DNA sequences.
14. The composition of matter of claim 1 , wherein the curing target sequence comprises the DNA sequence encoding the one or more editing gRNAs.
15. The composition of matter of claim 1 , wherein the one or more selectable markers are under the control of a constitutive promoter.
16. A library of vectors for recursive nucleic acid-directed CRISPR editing according to claim 1 .
17. The library of vectors of claim 16 , wherein the library of vectors comprises: (i) one or more DNA sequences encoding at least 1000 different editing gRNAs and (ii) one or more donor DNA sequences.
18. The library of vectors of claim 16 , further comprising a lambda Red recombineering system.
19. The composition of matter of claim 1 , wherein the one or more vectors further comprise a coding sequence for a nucleic acid-guided nuclease.
20. The composition of matter of claim 19 , wherein the coding sequence for the nucleic acid-guided nuclease is under the control of a third inducible promoter.
21. The composition of matter of claim 20 , wherein the first inducible promoter and the third inducible promoter are the same inducible promoter.
22. The composition of matter of claim 19 , wherein the nucleic acid-directed nuclease is a nuclease fusion enzyme.
23. The composition of matter of claim 1 , wherein the curing target sequence comprises a selectable marker.
24. The composition of matter of claim 1 , wherein the one or more vectors further comprise a lambda Red recombineering system.
25. The composition of matter of claim 1 , wherein the DNA sequence encoding a curing gRNA sequence, the DNA sequence encoding one or more editing gRNAs, and the one or more donor DNA sequences are on the same vector.
26. The composition of matter of claim 1 , wherein the DNA sequence encoding a curing gRNA sequence, the DNA sequence encoding one or more editing gRNAs, and the one or more donor DNA sequences are on two or more different vectors.