Cas9 gRNA engineering for genome editing, activation and repression

Abstract

We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

Figure 1

Activation and cutting of endogenous genes in HEK293T cells. (a–c) RNA expression and mutagenesis analysis of the genes ACTC1 (a), MIAT (b) and TTN (c). Each sample was transfected with the indicated Cas9 construct and gRNA of a particular length. Points represent data from two individual transfections. (d) Multiplexed activation and cutting of ACTC1, MIAT and TTN. The constructs were transfected with 20-nt ACTC1 gRNA and 14-nt MIAT and TTN gRNAs simultaneously. Points represent data from two individual transfections.

Figure 2

Design and experimental analysis of human cells with multifunctional CRISPR devices and circuits. (a) Top, schematic of a repression device using Cas9-VPR and 14-nt gRNA. Bottom, the geometric mean and s.d. of EYFP expression for cells expressing >10⁷ molecules of equivalent fluorescein (MEFL) of transfection marker enhanced blue fluorescent protein (EBFP). n = 3 independent technical replicates from three experiments (n = 2 for Cas9-VPR + gRNA). (b) Top, schematic of parallel Cas9-VPR and14-nt gRNA–based transcriptional repression and activation devices in a single cell. Bottom, the geometric mean and s.d. of EYFP and tdTomato expression for cells expressing >10⁷ MEFL of transfection marker EBFP. n = 4 independent technical replicates combined from three experiments. (c) Top, schematic of a genetic kill switch designed to incorporate Cas9-VPR DNA cleavage and transcriptional activation functions. Bottom, the geometric mean and s.d. of EYFP expression for cells expressing >3 × 10⁷ MEFL of transfection marker EBFP. n = 3 independent technical replicates from three experiments (n = 2 for Cas9-VPR without doxycycline). rtTA, reverse tetracycline-controlled transactivator; iRFP, near-infrared fluorescent protein. (d) Left, genetic kill-switch circuit incorporating all three functions of Cas9-VPR: DNA cleavage, transcriptional activation and repression. Right, the geometric mean and s.d. of EYFP expression for cells expressing >10⁷ MEFL of transfection marker mKate. n = 4 independent technical replicates from three experiments (n = 2 in 48-h groups). pConst, constitutively active promoter (e.g., CAG or hEF1-a).