Crispr ribonucleoprotein complex genome editing of human innate immune cells

A method of electroporating a CRISPR ribonucleoprotein complex comprising from 40 to 100 pmol Cas9 complexed with from 120 to 300 pmol sgRNA into human CD14+ monocytes, the method comprising:

• collecting leucocytes from an individual;
• partially purifying the leucocytes to generate a population of cells enriched for CD14+ monocytes;
• electroporating the CD14+ monocytes within 24 hours of collection from the individual;
• combining the CRISPR ribonucleoprotein complex with the enriched population of cells;
• electroporating the CRISPR ribonucleoprotein complex that has been combined with the enriched population of cells under conditions comprising:
• a voltage between 1700-2000 volts;
• and
• a pulse width of width of about 1×20-30 milliseconds;
• such that the CRISPR ribonucleoprotein complex is electroporated into the CD14+ monocytes.

A method of electroporating a CRISPR ribonucleoprotein complex comprising from 40 to 100 pmol Cas9 complexed with from 120 to 300 pmol sgRNA into human CD34+ hematopoietic stem cells, the method comprising:

• collecting leucocytes from an individual;
• partially purifying the leucocytes to generate a population of cells enriched for CD34+ hematopoietic stem cells;
• culturing the CD34+ hematopoietic stem cells for at least 3 days prior to electroporation;
• combining the CRISPR ribonucleoprotein complex with the enriched population of cells;
• electroporating the CRISPR ribonucleoprotein complex that has been combined with the enriched population of cells under conditions comprising:
• a voltage between 1700-2000 volts;
• and
• a pulse width of width of about 1×20-30 milliseconds;
• such that the CRISPR ribonucleoprotein complex is electroporated into the CD34+ hematopoietic stem cells.

A method of electroporating a CRISPR ribonucleoprotein complex comprising from 40 to 100 pmol Cas9 complexed with from 120 to 300 pmol sgRNA into human CD56+ Natural Killer cells, the method comprising:

• collecting leucocytes from an individual;
• partially purifying the leucocytes to generate a population of cells enriched for CD56+ Natural Killer cells;
• culturing the CD56+ Natural Killer cells for at least 3-17 days prior to electroporation;
• combining the CRISPR ribonucleoprotein complex with the enriched population of cells;
• electroporating the CRISPR ribonucleoprotein complex that has been combined with the enriched population of cells under conditions comprising:
• a voltage between 1700-2000 volts;
• and
• a pulse width of width of about 1×20-30 milliseconds;
• such that the CRISPR ribonucleoprotein complex is electroporated into the CD56+ Natural Killer cells.