Compositions, methods, modules and instruments for automated nucleic acid-guided nuclease editing in mammalian cells using microcarriers

Abstract

Compositions of matter, methods, modules, and automated instruments may relate to synthesizing a library including an editing cassette including a different gRNA and donor DNA pair, amplifying the editing cassette in a partition separate from other editing cassettes in the library, adding nuclease to the partition, and adding lipofectamine to the editing cassette and nuclease to form a lipofectamine/nucleic acid/nuclease complex. A microcarrier coated in extracellular matrix or a cell adhesion molecule coating may be added to the lipofectamine/nucleic acid/nuclease complex. Cell growth material, the microcarrier, and mammalian cells may be transferred to a growth module in an automated closed cell editing instrument via a liquid handling system. The mammalian cells may be allowed to seed on the microcarrier. Conditions may be provided for the mammalian cells to take-up and be edited by a payload associated with the lipofectamine/nucleic acid/nuclease complex. The mammalian cells may be detached from the microcarrier.

1 . A method for transfecting and performing nucleic acid-guided nuclease editing in mammalian cells in an automated closed cell editing instrument comprising the steps of comprising the steps of:
 synthesizing a library of editing cassettes off instrument, wherein each editing cassette comprises a different gRNA and donor DNA pair;   amplifying each editing cassette in the library of editing cassettes in a partition separate from other editing cassettes;   adding nuclease to each partition with an amplified editing cassette;   adding lipofectamine to each amplified editing cassette and nuclease to form a library of lipofectamine/nucleic acid/nuclease complexes;   adding microcarriers to each lipofectamine/nucleic acid/nuclease complex in the library of lipofectamine/nucleic acid/nuclease complexes, wherein the microcarriers are coated in extracellular matrix or a cell adhesion molecule coating and wherein the lipofectamine/nucleic acid/nuclease complexes bind to the microcarriers;   transferring cell growth medium, the microcarriers and mammalian cells to a growth module in the automated closed cell editing instrument via a liquid handling system;   allowing the cells to seed on the coated microcarriers in the growth module;   providing conditions for the cells to take-up the lipofectamine/nucleic acid/nuclease payloads in the growth module;   providing conditions for the nucleic acids and nuclease to edit the cells in the growth module; and   detaching the edited cells from the microcarriers.   
     
     
         2 - 30 . (canceled)