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AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology
InventorsFeng Zhang, Makoto Saito, Guilhem Faure
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Systems and methods for targeted gene modification, targeted insertion, perturbation of gene transcripts, and nucleic acid editing. Novel nucleic acid targeting systems comprise components of CRISPR systems and transposable elements. CRISPR-associated transposons (CASTs) are bacterial RNA-guided Tn7-like transposons that can insert large DNA cargoes at targeted loci in bacteria. To adapt CAST for use on the human genome, Applicants screened 30 CAST I-B systems for activity on extrachromosomal DNA in human cells. Of the six active orthologs identified, Applicants engineered a I-B2 system from Tolypothrix sp. PCC7910 (TolCAST) to achieve RNA-guided DNA insertion in the genome of living human cells.
An engineered system, the system comprising:
The engineered system of any of the previous claims, wherein the insertion is about 80 to 100 base pairs, more preferably 80 to 95 nucleotides, after PAM of the target polynucleotide.
The engineered system of any of the previous claims, wherein the Type I-B Cas protein is IB-43.
The engineered system of any of the previous claims, wherein the one or more CRISPR-associated Tn7 or Tn7-like transposase polypeptides comprise TnsA, TnsB, TnsC, and/or TniQ.
The engineered system of any of the previous claims, wherein the one or more Type I-B Cas proteins comprises Cas5, Cas6, Cas7, Cas 8, Cas11, or a combination thereof.
The engineered system of any of the previous claims, wherein the one or more Type I-B Cas proteins comprises a Cas11.
The engineered system of any of the previous claims further comprising a ClpX.
The engineered system of claim any of the previous claims, wherein each of the one or more Tn7 transposase polypeptides is independently derived from a same or different species.
The engineered system of claim any of the previous claims, wherein each of the one or more Type I-B Cas proteins is derived from a same or different species.
The engineered system of any of the previous claims, further comprising a donor polynucleotide.
The engineered system of any of the previous claims, further comprising a nuclear localization signal (NLS).
The system of anyone of the preceding claims, wherein a, b, c, or a combination thereof is encoded by a one or more polynucleotides.
A method of inserting a donor polynucleotide into a target polynucleotide in a cell, the method comprises introducing to the cell:
Dependencies are inferred from the claim text.