Type V CRISPR-Cas systems and use thereof
Engineered or non-naturally occurring systems and compositions comprising a novel Cas12b and a guide molecule. Also provided include methods of use the systems and compositions, including in treating and diagnosing diseases.
1. A non-naturally occurring or engineered composition comprising
a Cas12b protein from Alicyclobacillus acidiphilus ; and
a guide molecule that does not impede the Cas12b's activity at elevated temperatures above 55° C., comprising a guide sequence with at least 95% or more sequence similarity to a direct repeat and tracrRNA sequence from the Alicyclobacillus acidoterrestris CRISPR-Cas12b system and capable of forming a complex with the Cas12b protein and directing the complex to bind to a target polynucleotide.
2. The composition of claim 1 , wherein the guide sequence comprises one of SEQ ID NOs: 61957-61961.
3. The composition of claim 1 , wherein the Cas12b protein is fused to one or more localization signals.
4. The composition of claim 1 , further comprising a detection construct comprising a non-target polynucleotide, wherein the Cas protein exhibits collateral activity and cleaves the non-target polynucleotide component once activated by the target polynucleotide.
5. The composition of claim 4 , further comprising one or more isothermal amplification reagents.
6. The composition of claim 5 , wherein the isothermal amplification reagents are loop-mediated isothermal amplification (LAMP) reagents, wherein the LAMP reagents are primers selected from SEQ ID NOs: 61983-61988.
7. The composition of claim 6 , wherein the guide molecule is designed to bind to a polynucleotide sequence of SARS-CoV-2, wherein the guide molecule is SEQ ID NO: 61989.
8. A vector composition comprising one or more polynucleotide sequences encoding the Cas12b protein and the guide molecule in the composition of claim 1 .
9. A non-naturally occurring or engineered prokaryotic or eukaryotic isolated host cell comprising the composition of claim 1 , or progeny thereof.
10. A method of targeting one or more target polynucleotides, the method comprising contacting the one or more target polynucleotides with a non-naturally occurring or engineered composition of claim 1 , wherein targeting comprises modifying the one or more target polynucleotides comprises increasing or decreasing expression of the one or more genes in the one or more target polynucleotides, insertion of a recombination template or a portion thereof to the one or more target polynucleotides.
11. A method for detecting a target polynucleotide in a sample, comprising
contacting the sample with the composition of claim 4 , wherein the Cas protein exhibits collateral activity and cleaves the detection construct once activated by the target polynucleotide, and the cleaved detection construct generate a signal; and
detecting the signal thereby determining presence of the target polynucleotide in the sample.
12. A kit for modifying or detecting a target polynucleotide in a sample, comprising the composition of claim 1 .
13. The composition of claim 1 , wherein the guide molecule comprises a sequence with at least 95% or more sequence similarity to one of SEQ ID NOs: 61957-61961.
14. The vector composition of claim 8 , wherein the polynucleotide sequences are codon optimized for expression in a eukaryotic cell.