Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis

Abstract

Genome engineering in human pluripotent stem cells (hPSCs) holds great promise for biomedical research and regenerative medicine. Recently, an RNA-guided, DNA-cleaving interference pathway from bacteria [the type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) pathway] has been adapted for use in eukaryotic cells, greatly facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus pyogenes and Streptococcus thermophilus), each with their own distinct targeting requirements and limitations, have been developed for genome editing thus far. Furthermore, limited information exists about homology-directed repair (HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these systems. Here, using a distinct CRISPR-Cas system from Neisseria meningitidis, we demonstrate efficient targeting of an endogenous gene in three hPSC lines using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9) recognizes a 5'-NNNNGATT-3' protospacer adjacent motif (PAM) different from those recognized by Cas9 proteins from S. pyogenes and S. thermophilus (SpCas9 and StCas9, respectively). Similar to SpCas9, NmCas9 is able to use a single-guide RNA (sgRNA) to direct its activity. Because of its distinct protospacer adjacent motif, the N. meningitidis CRISPR-Cas machinery increases the sequence contexts amenable to RNA-directed genome editing.

Keywords: crRNA; embryonic stem cells; induced pluripotent stem cells; tracrRNA.

Fig. 1.

Functional NmCas9 can be expressed in mammalian cells. (A) Western blot analysis demonstrates that FLAG-tagged NmCas9 is expressed in 293FT cells. (Lane 1) Untransfected 293FT cells. (Lane 2) 293FT cells transfected with FLAG-tagged NmCas9 expressing plasmid. (Upper) Anti-FLAG tag Western. (Lower) Anti-GAPDH Western as loading control. (B) Design of the crRNA that targets the tdTomato coding sequence. Blue: PAM sequence; orange: crRNA spacer; green: crRNA repeat. (C) The plasmid containing the tdTomato coding sequence (see B) was linearized with NdeI and mixed with different combinations of tracrRNA, crRNA, and cell lysate prepared from NmCas9-expressing 293FT cells. After incubation at 37 °C, DNA was purified and analyzed by agarose gel electrophoresis. The diagram on the right shows the expected cleavage products and their predicted sizes. “N” indicates inclusion of a nonspecific crRNA that does not target tdTomato. (D) Cleavage product (see C) was extracted from the gel and analyzed by Sanger sequencing using the primers indicated in the Right panel. The cleavage site, indicated by the arrow, was inferred from the sequencing.

Fig. 2.

NmCas9 functions in gene disruption in human ES cells. (A–C) The localization of NmCas9 with an NLS at the N terminus (A), C terminus (B), or both termini (C) was analyzed by either EGFP fluorescence (A) or anti-HA immunofluorescence (B and C) in 293FT cells. (Scale bars, 20 µm.) (D) The localization of NmCas9 with the double NLS (see C) was analyzed by anti-HA immunofluorescence in hESCs. (Scale bars, 20 µm.) (E) Design of a single plasmid used for gene editing in hESCs. (F) FACS analysis of tdTomato reporter hESC lines after electroporation of the indicated crRNA/tracrRNA/NmCas9 constructs. The number in the plot indicates the percentage of tdTomato-fluorescence-negative cells 5 d after electroporation. (G) Indels introduced by the targeting CRISPR in the tdTomato negative population (see F) were analyzed by targeted PCR amplification and sequencing. The protospacer sequence is underlined. The numbers in parentheses indicate the number of sequenced clones containing that specific indel.

Fig. 3.

Specificity screen of NmCas9 system. (A, Upper) The crRNA sequence targeting tdTomato, with locations of single point mutations (black) in the spacer region of the tdTomato targeting crRNA. (Lower) The efficiency of each mutant at disrupting tdTomato expression. All mutants were tested as described in Fig. 2F. The efficiency is defined as percent tdTomato-negative cells (mutant spacer) divided by percent tdTomato-negative cells (wild-type spacer). Error bars: SEM (B, Upper) The locations of different mutant PAMs in the tdTomato sequence. Blue, wild-type PAM; brown, mutant PAM; underlined, spacer sequence. For the bottom-most protospacer, the opposite strand was targeted, and the reverse complement sequence is therefore shown. (Lower) The efficiency of targeting at each site associated with the indicated PAM, as revealed by the loss of tdTomato expression. All targeting experiments were performed as described in Fig. 2F. The efficiency is defined as percent tdTomato-negative cells (mutant PAM) divided by percent tdTomato-negative cells (wild-type PAM). Error bars: SEM.

Fig. 4.

Gene targeting in hESCs using NmCas9. (A) Donor DNA and crRNA design. The mismatch in the first nucleotide of crRNA is to satisfy the requirement of the U6 promoter for a G residue at the transcription start site. (B) Phase-contrast (Upper) and fluorescent (Lower) images of targeted clones from H1, H9, and iPS005 line. (Scale bars, 50 µM.) (C) Southern blot analysis of H1 WT (lane 1), H9 WT (lane 3), and iPS005 WT (lane 5) and targeted clones of H1 (lane 2), H9 (lane 4), and iPS005 (lane 6) line. Genomic DNA was digested with BamHI. The Southern probe is located outside of donor DNA (see A). The wild-type clone should give one band of 4.2 kb and targeted heterozygous clone should give one additional band of 5.6 kb. (D) Targeted clones (see B) were treated with 10 µM SB431542 and 10 ng/mL BMP4 to initiate differentiation. The EGFP signal was analyzed by FACS 3 d after differentiation. Gray, undifferentiated parental cells before targeting; green, targeted cells before differentiation; blue, targeted cells after differentiation.