Compositions, systems, and methods for generating gene-edited cells

A method of generating a gene-edited stem or progenitor cell comprising:

• a) contacting an initial cell from a subject with first, second, third, fourth, and fifth reagents in vitro,
• wherein said initial cell comprises a mutation in its genome that is responsible for a disease or condition in a subject from which cell was derived,
• wherein said first reagent comprises a first mRNA encoding a first protein, wherein said first protein is capable of reducing, suppressing or preventing an innate immune response in said initial stem or progenitor cell,
• wherein said second reagent comprises a second mRNA encoding a second protein that inhibits 53BPI,
• wherein said third reagent comprises a third mRNA encoding a third protein, wherein
• said third protein comprises a Cas nuclease for use in CRISPR,
• wherein said fourth reagent comprises a guide RNA (gRNA or sgRNA) comprising a tracrRNA having a sequence that binds to a Cas enzyme and a CRISPR RNA (crRNA) having a short “guide” sequence that is complementary to and binds to a specific target cleavage site in the genome that is near to said mutation, and
• wherein said fifth reagent comprises a donor oligodeoxynucleotide (donor ODN), wherein said ODN comprises two outer flanking regions that are complementary to one strand of the genomic DNA on each side of the mutation site region in said genomic DNA and a middle region of interest, wherein said region of interest is configured to replace said disease/condition-causing mutation region in said genomic DNA by homology-directed repair (HDR) after said genomic DNA is cleaved by said CRISPR-Cas nuclease at said target cleavage site,
• wherein said contacting is under conditions such that said region of interest replaces said disease/condition-causing mutation region in said genomic DNA, thereby generating a gene-edited cell.

A composition comprising:

• a) mRNA encoding a first protein, wherein said first protein is capable of reducing, suppressing or preventing an innate immune response in a human or animal cell, and
• wherein a mutation in the genome of said cell is responsible for a disease or condition in a human or animal subject;
• b) a second mRNA encoding a second protein that inhibits 53BPI,
• c) mRNA encoding a third protein, wherein said third protein comprises a Cas nuclease for CRISPR;
• and
• d) guide RNA (gRNA or sgRNA) comprising a tracrRNA having a sequence that binds to a Cas enzyme and a CRISPR RNA (crRNA) having a short “guide” sequence that is complementary to and binds to a specific target cleavage site in the genome that is near to said mutation.