High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening

Rodrigo A Gier; Krista A Budinich; Niklaus H Evitt; Zhendong Cao; Elizabeth S Freilich; Qingzhou Chen; Jun Qi; Yemin Lan; Rahul M Kohli; Junwei Shi

doi:10.1038/s41467-020-17209-1

High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening

Nat Commun. 2020 Jul 13;11(1):3455. doi: 10.1038/s41467-020-17209-1.

Authors

Rodrigo A Gier^#^{1

2

3

4}, Krista A Budinich^#^{1

2

3}, Niklaus H Evitt^{1

2

3

5}, Zhendong Cao^{1

2

3}, Elizabeth S Freilich^{1

2

3}, Qingzhou Chen^{1

2

3}, Jun Qi⁶, Yemin Lan², Rahul M Kohli^{2

5}, Junwei Shi^{7

8

9}

Affiliations

¹ Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
² Epigenetics Institute, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
³ Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
⁴ Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, 19104, USA.
⁵ Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
⁶ Department of Cancer Biology, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, Boston, MA, 02215, USA.
⁷ Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. jushi@upenn.edu.
⁸ Epigenetics Institute, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. jushi@upenn.edu.
⁹ Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. jushi@upenn.edu.

^# Contributed equally.

Abstract

CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Bacterial Proteins / genetics*
CRISPR-Associated Proteins / genetics*
CRISPR-Cas Systems*
Cell Proliferation
Endodeoxyribonucleases / genetics*
Epigenesis, Genetic
Gene Editing*
Gene Expression Regulation, Leukemic*
Gene Library
Genetic Engineering
Genome, Human
HEK293 Cells
Humans
K562 Cells
Leukemia / genetics*
Mice
NIH 3T3 Cells
Protein Domains
RNA, Guide, CRISPR-Cas Systems / genetics

Substances

Bacterial Proteins
CRISPR-Associated Proteins
RNA, Guide, CRISPR-Cas Systems
Cas12a protein
Endodeoxyribonucleases

Grants and funding

R01 GM118501/GM/NIGMS NIH HHS/United States