In vivo combinatorial knockout screens using CRISPR-Cpf1

Abstract

Metastasis is the major lethal factor of solid cancers. However, the genetic interactions that influence metastatic potential have been challenging to investigate in a systematic manner. A streamlined method for mapping genetic interactions in vivo is key to understanding metastasis at the systems level. Here we developed MCAP (massively-parallel CRISPR-Cpf1 crRNA array profiling), an approach for combinatorial interrogation of double knockouts in vivo. We designed an MCAP library targeting 325 pairwise combinations of genes implicated in metastasis, and functionally interrogated their metastatic potential in mice. This protocol accompanies Chow et al. Nature Methods, “In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens”. Introduction CRISPR screens are a powerful approach to systematically identify the genetic factors involved in a biological function of interest 1. While the vast majority of CRISPR screens to date have focused on the contributions of individual genes, many biological phenomena cannot be fully captured at the single-gene level due to existence of genetic interactions 2,3. A number of recent studies have demonstrated the use of double-knockout/perturbation CRISPR-Cas9 screens to characterize these genetic interactions 4–9. However, current Cas9-based approaches for mapping genetic interactions have important limitations that hinder their broader utility and applicability. Towards this end, Cpf1 (also known as Cas12a) has notable advantages over Cas9, as Cpf1 can autonomously process a single array containing multiple crRNAs and subsequently induce multi-site mutagenesis 10–13. In the associated publication, we describe an approach for combinatorial knockout screens using massively-parallel CRISPR-Cpf1 crRNA array profiling (MCAP). Compared to prior Cas9-based combinatorial screens, MCAP is unparalleled in its simplicity of oligo design, library cloning, and downstream analysis, thereby facilitating its application to the study of complex processes in vivo. Figure 1 illustrates an example MCAP workflow to identify genetic interactions driving metastasis. We anticipate the MCAP system to be of use to the scientific community for the study of two-gene or even higher-order genetic interactions. Figure 1. Schematics for massively-parallel CRISPR-Cpf1 crRNA array profiling (MCAP) to interrogate metastasis driver combinations in vivo Reagents and Equipment Plasmids & DNA 1. Plasmids: pRC10 (pLenti-EFS-huLbCpf1-2A-Blast-WPRE; Addgene #123359), pRC11 (pLentiU6-DR-crRNA-BsmbI(x2)/EFS-Puro-WPRE; Addgene #123360), pRG01 (pLenti-U6-DRcrRNA-BsmbI(x2)-6T/EFS-Puro-2A-Fluc-WPRE; Addgene #123362), pRG01-MCAP_Met (pLenti-U6-MCAP_Met_lib/EFS-Puro-2A-Fluc-WPRE; Addgene #123361), pRC49 (pLenti-U6DR-crRNA-BsmbI(x2)-6T/EFS-Puro-2A-Fluc-2A-EGFP_NLS-WPRE; Addgene #123363), pMD2.G (Addgene #12259), psPAX.2 (Addgene #12260), pcDNA3-EGFP (Addgene #13031) 2. Cpf1 crRNA oligos: DNA oligos to clone into the lentiviral crRNA expression vectors, see below for details on oligo design for library-scale or single-construct cloning 3. Readout primers: DNA oligos to sequence the U6 expression cassettes and readout library-wide crRNA representation