Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

Xander Nuttle; Nicholas D Burt; Benjamin Currall; Mariana Moysés-Oliveira; Kiana Mohajeri; Riya Bhavsar; Diane Lucente; Rachita Yadav; Derek J C Tai; James F Gusella; Michael E Talkowski

doi:10.1016/j.crmeth.2023.100672

Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

Cell Rep Methods. 2024 Jan 22;4(1):100672. doi: 10.1016/j.crmeth.2023.100672. Epub 2023 Dec 12.

Affiliations

¹ Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Department of Neurology, Harvard Medical School, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA. Electronic address: anuttle@mgh.harvard.edu.
² Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA.
³ Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Department of Neurology, Harvard Medical School, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA.
⁴ Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA; PhD program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA.
⁵ Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA; Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA.
⁶ Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Department of Neurology, Harvard Medical School, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA. Electronic address: mtalkowski@mgh.harvard.edu.

Abstract

New technologies and large-cohort studies have enabled novel variant discovery and association at unprecedented scale, yet functional characterization of these variants remains paramount to deciphering disease mechanisms. Approaches that facilitate parallelized genome editing of cells of interest or induced pluripotent stem cells (iPSCs) have become critical tools toward this goal. Here, we developed an approach that incorporates libraries of CRISPR-Cas9 guide RNAs (gRNAs) together with inducible Cas9 into a piggyBac (PB) transposon system to engineer dozens to hundreds of genomic variants in parallel against isogenic cellular backgrounds. This method empowers loss-of-function (LoF) studies through the introduction of insertions or deletions (indels) and copy-number variants (CNVs), though generating specific nucleotide changes is possible with prime editing. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of isogenic cellular collections and catalyze comparative functional genomic studies investigating the roles of hundreds of genes and mutations in development and disease.

Keywords: CP: Genetics; CP: Stem cell; CRISPR; allelic series; functional genomics; gRNA library; genome editing; genome engineering; induced pluripotent stem cells; mutational modeling; piggyBac transposon.

MeSH terms

CRISPR-Cas Systems*
Gene Editing / methods
Genomics
Humans
Induced Pluripotent Stem Cells*
Mutation

Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

Authors

Affiliations

Abstract

MeSH terms

Grants and funding