Methods and compositions comprising CRISPR-Cpf1 and paired guide CRISPR RNAs for programmable genomic deletions

Inventors

Sanjana, Neville Espi

Assignees

New York University • New York Genome Center

Abstract

Described are methods comprises transducing a mammalian cell with one or more virus vectors. Each vector comprises a nucleic acid sequence encoding a Cpf1 (also known as Cas12a) protein and an optional selectable marker in operative association with an RNA pol II promoter which controls expression thereof, and a CRISPR RNA (crRNA) array comprising at least two spacers in operative association with an RNA pol III promoter. Each spacer encodes an RNA guide which hybridizes to a unique sequence located 3′ from a T-rich protospacer-adjacent motif (PAM) in a genomic region of interest. The method further comprises culturing the transduced cells, thereby providing a plurality of cultured cell cultures, each cell culture comprising said deletion. Additionally, described are compositions used in methods as well as libraries generated by the methods. Such compositions comprise libraries of transduced cell cultures, viral vectors, nucleic acid sequences, CRISPR RNA spacers, and RNA guides, as described herein.

Core Innovation

The invention provides a CRISPR-Cpf1 programmable genomic deletion system in which mammalian cells are transduced with one or more lentiviral vectors. Each vector comprises a nucleic acid sequence encoding a Cpf1 (Cas12a) protein in operative association with an RNA pol II promoter that controls expression in a mammalian cell, and also comprises a flipped CRISPR RNA (crRNA) array having at least two spacers, wherein each spacer encodes an RNA guide and the array is in operative association with an RNA pol III promoter.

The crRNA guides are configured such that each guide hybridizes to a unique sequence located 3' from a T-rich protospacer-adjacent motif (PAM) in a contiguous region of a genome or a chromosome of a mammalian cell. In the transduced and cultured cells, Cpf1 (Cas12a) creates a deletion comprising the chromosome or genome between cleavage sites located downstream of the PAM, thereby providing a plurality of transduced cell cultures, each cell culture comprising the respective deletion.

The invention supports programmable deletion libraries by using a crRNA array architecture in operative association with an RNA pol III promoter and an independently controlled RNA pol II promoter in opposing orientations. It also contemplates selection and library generation of deletion-containing cell cultures, including deletions in contiguous genomic regions and deletion geometries defined by cleavage-site downstream spacing.

Claims Coverage

Independent claim clm-00001 covers an in vitro method that uses lentiviral vector transduction of mammalian cells with RNA pol II-driven Cpf1 (Cas12a) and an opposing-orientation flipped crRNA array under RNA pol III control, producing multiple deletion-containing transduced cell cultures. The claim includes four inventive features.

Claim clm-00001 covers a lentiviral, programmable Cpf1 (Cas12a) deletion method in mammalian cells where Pol II controls Cpf1 expression and an independent opposing-orientation Pol III drives a flipped multi-spacer crRNA array; the crRNA guides target unique 3' positions relative to T-rich PAMs, and Cpf1 generates deletions between downstream cleavage sites to yield multiple transduced cell cultures containing the deletions.

Stated Advantages

Documented Applications