Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 May 12;11(6):875-883.
doi: 10.1016/j.celrep.2015.04.007. Epub 2015 Apr 30.

Efficient CRISPR-Cas9-mediated Generation of Knockin Human Pluripotent Stem Cells Lacking Undesired Mutations at the Targeted Locus

Affiliations
Free PMC article

Efficient CRISPR-Cas9-mediated Generation of Knockin Human Pluripotent Stem Cells Lacking Undesired Mutations at the Targeted Locus

Florian T Merkle et al. Cell Rep. .
Free PMC article

Abstract

The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.

Figures

Figure 1
Figure 1. CRISPR-Cas9 mediated gene targeting efficiency in hPSCs
(A) Percentage of analyzed drug-resistant clones that gave PCR products of the expected size across both the 5′ and 3′ homology arms for each locus and Cas9 targeting strategy tested. Each dot represents a distinct combination of CRISPR sequence and targeted locus for a given Cas9 strategy. Clone numbers and targeting efficiencies are listed in Table 1. (B) Summary of targeting efficiencies for each Cas9 strategy at each locus and CRISPR combination (left) or at distinct genes (right). (C) Relative targeting efficiency at the same locus and guide sequence but with different Cas9 enzymes. (D) Relationship between number of clones analyzed and number of clones correctly targeted for each Cas9-based strategy. Error bars in B, and C denote SEM. See also Figure S1.
Figure 2
Figure 2. Frequency of CRISPR-Cas9 induced on-target indels
(A) Schematic of a correctly targeted human pluripotent stem cell clone showing untargeted and targeted alleles, CRISPR-Cas9 cleavage sites (triangles) and the sequenced amplicons. (B) Schematic of deep sequencing data, showing the genomic locus with the CRISPR guide sequence (red), PAM motif (blue), predicted cleavage site (triangles), and the calculation of the indel frequency. (C) The indel frequency at the untargeted allele of correctly targeted hPSC clones (number of sequenced clones indicated) is plotted for each targeted locus and Cas9 strategy. (D) Mean indel frequencies at the untargeted allele of correctly targeted hPSC clones. (E,F) Analysis as in (C,D) but performed at targeted alleles with intact CRISPR target sites. See also Fig. S2. (G) Number of intact and unique indel alleles (sub-clones) per correctly targeted hPSC clone, examined at the untargeted allele HCRT-C locus. (H) Average allele frequency (sub-clone size) in hPSC clones. (I) Schematic diagram illustrating the hypothesis of heterogeneous clone formation. Error bars in D, F, and H denote SEM. See also Figure S2.
Figure 3
Figure 3. Strategy for generating intact knock-in hPSC lines
(A) Schematic of a prototypical targeted allele with CRISPR target sites physically separated by gene insertion. Cas9dn cleavage sites (triangles), 5′ amplicon (T5′) and 3′ amplicon (T3′) across 5′ and 3′ CRISPR target sites of the Cas9dn strategy are illustrated. (B,C) Indel frequencies at the targeted allele of Cas9dn-targeted clones shown for each clone (B) or as the mean indel frequency for each locus (C). (D) Mean indel frequencies at the untargeted allele of correctly targeted hPSC clones. (E) Percentage of clones with indel frequencies of 2% or less at the untargeted allele plotted against targeting efficiency for each locus. (F) Percentage of correctly targeted clones that are intact at both the targeted and untargeted allele. (G) Percentage of correctly targeted and intact clones among all drug-resistant clones screened. (H) Workflow for identifying clones of knock-in hPSC clones free of unwanted mutations at the targeted locus. Error bars in D, F, and G denote SEM. See also Figure S3.

Similar articles

See all similar articles

Cited by 50 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback