RNA-guided human genome engineering via Cas9

Abstract

Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

Fig. 1. Genome editing in human cells using an engineered type II CRISPR system

(A) RNA-guided gene targeting in human cells involves coexpression of the Cas9 protein bearing a C-terminal SV40 nuclear localization signal (NLS) with one or more gRNAs expressed from the human U6 polymerase III promoter. Cas9 unwinds the DNA duplex and cleaves both strands upon recognition of a target sequence by the gRNA, but only if the correct PAM is present at the 3′ end. Any genomic sequence of the form GN₂₀GG can, in principle, be targeted. CMV, cytomegalovirus promoter; TK, thymidine kinase; pA, polyadenylation signal. (B) A genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68-bp genomic fragment from the AAVS1 locus. Restoration of the GFP sequence by HR with an appropriate donor sequence results in GFP⁺ cells that can be quantified by FACS. T1 and T2 gRNAs target sequences within the AAVS1 fragment. Binding sites for the two halves of the TALEN are underlined. (C) Bar graph depicting HR efficiencies induced by T1, T2, and TALEN-mediated nuclease activity at the target locus, as measured by FACS. Representative FACS plots and microscopy images of the targeted cells are depicted below. (Scale bar, 100 μm.) Data are shown as means ± SEM (N = 3).

Fig. 2. RNA-guided genome editing of the native AAVS1 locus in multiple cell types

(A) T1 (red) and T2 (green) gRNAs target sequences in an intron of the PPP1R12C gene within the chromosome 19 AAVS1 locus. (B) Total count and location of deletions caused by NHEJ in 293Ts, K562s, and PGP1 iPS cells after expression of Cas9 and either T1 or T2 gRNAs as quantified by next-generation sequencing. Red and green dashed lines demarcate the boundaries of the T1 and T2 gRNA targeting sites. NHEJ frequencies for T1 and T2 gRNAs were 10% and 25% in 293T, 13% and 38% in K562, and 2% and 4% in PGP1 iPS cells, respectively. (C) DNA donor architecture for HR at the AAVS1 locus, and the locations of sequencing primers (arrows) for detecting successful targeted events, are depicted. (D) PCR assay 3 days after transfection demonstrates that only cells expressing the donor, Cas9 and T2 gRNA exhibit successful HR events. (E) Successful HR was confirmed by Sanger sequencing of the PCR amplicon, which showed that the expected DNA bases at both the genome-donor and donor-insert boundaries are present. (F) Successfully targeted clones of 293T cells were selected with puromycin for 2 weeks. Microscope images of two representative GFP+ clones is shown. (Scale bar, 100 μm.)