Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System

Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease

Hierarchical and stage-specific regulation of murine cardiomyocyte maturation by serum response factor

After birth, cardiomyocytes (CM) acquire numerous adaptations in order to efficiently pump blood throughout an animal’s lifespan. How this maturation process is regulated and coordinated is poorly understood. Here, we perform a CRISPR/Cas9 screen in mice and identify serum response factor (SRF) as a key regulator of CM maturation. Mosaic SRF depletion in neonatal CMs disrupts many aspects of their maturation, including sarcomere expansion, mitochondrial biogenesis, transverse-tubule formation, and cellular hypertrophy. Maintenance of maturity in adult CMs is less dependent on SRF. This stage-specific activity is associated with developmentally regulated SRF chromatin occupancy and transcriptional regulation. SRF directly activates genes that regulate sarcomere assembly and mitochondrial dynamics. Perturbation of sarcomere assembly but not mitochondrial dynamics recapitulates SRF knockout phenotypes. SRF overexpression also perturbs CM maturation. Together, these data indicate that carefully balanced SRF activity is essential to promote CM maturation through a hierarchy of cellular processes orchestrated by sarcomere assembly

Host non-inflammatory neutrophils mediate the engraftment of bioengineered vascular networks

Notwithstanding remarkable progress in vascular network engineering, implanted bioengineered microvessels largely fail to form anastomoses with the host vasculature. Here, we demonstrate that implants containing assembled human vascular networks (A-Grafts) fail to engraft due to their inability to engage non-inflammatory host neutrophils upon implantation into mice. In contrast, unassembled vascular cells (U-Grafts) readily engage alternatively polarized neutrophils, which in turn serve as indispensable mediators of vascular assembly and anastomosis. The depletion of host neutrophils abrogated vascularization in U-Grafts, whereas an adoptive transfer of neutrophils fully restored vascularization in myeloid-depleted mice. Neutrophil engagement was regulated by secreted factors and was progressively silenced as the vasculature matured. Exogenous addition of factors from U-Grafts reengaged neutrophils and enhanced revascularization in A-Grafts, a process that was recapitulated by blocking Notch signaling. Our data suggest that the pro-vascularization potential of neutrophils can be harnessed to improve the engraftment of bioengineered tissues

Hierarchical and stage-specific regulation of murine cardiomyocyte maturation by serum response factor

After birth, cardiomyocytes (CM) acquire numerous adaptations in order to efficiently pump blood throughout an animal’s lifespan. How this maturation process is regulated and coordinated is poorly understood. Here, we perform a CRISPR/Cas9 screen in mice and identify serum response factor (SRF) as a key regulator of CM maturation. Mosaic SRF depletion in neonatal CMs disrupts many aspects of their maturation, including sarcomere expansion, mitochondrial biogenesis, transverse-tubule formation, and cellular hypertrophy. Maintenance of maturity in adult CMs is less dependent on SRF. This stage-specific activity is associated with developmentally regulated SRF chromatin occupancy and transcriptional regulation. SRF directly activates genes that regulate sarcomere assembly and mitochondrial dynamics. Perturbation of sarcomere assembly but not mitochondrial dynamics recapitulates SRF knockout phenotypes. SRF overexpression also perturbs CM maturation. Together, these data indicate that carefully balanced SRF activity is essential to promote CM maturation through a hierarchy of cellular processes orchestrated by sarcomere assembly

Effect of homology arm length for Cas9-mediated knockin.

<p><b>A.</b> GFP knockin at Oct4 C-terminus using 50 bp or 200 bp homology arms. Knockin efficiency was measured by FACS for GFP expression. <b>B–C.</b> GFP expression in GFP⁺ FACS clones by microscopy and western blotting. <b>D.</b> PCR genotyping of individual ESC clones. The PCR product (arrowhead) is diagnostic of knockin at <i>Oct4</i>.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Knockout of 3 congenital heart disease genes in mESCs.

<p><b>A.</b> Experimental outline. Paired gRNAs are designed to induce a deletion of about 100 bp. <b>B.</b> PCR genotyping of pooled ESC genomic DNA after <i>Smad2</i> targeting. The mutant PCR product predominated. <b>C.</b> Surveyor nuclease on pooled ESC genomic DNA. Red arrowheads indicate nuclease cleavage products indicative of heterodimers. <b>D.</b> PCR genotyping of individual clones showing Smad2^Δ/Δ, Smad2^+/+, and Smad2^+/Δ clones. <b>E–F.</b> Frequency of <i>Smad2</i> genotypes amongst clonal outgrowths, after enrichment for transfected cells by GFP FACS or by transient puromycin selection. <b>G.</b> Western blot measurement of <i>Smad2</i> expression in wild-type and Smad2^Δ/Δ clones. <b>H.</b> Frequency of <i>Mll2</i> genotypes amongst 39 genotyped clones. <b>I.</b> Confirmation of <i>Mll2</i> inactivation in individual Mll2^Δ/Δ clones by qRTPCR. <b>J.</b> Frequency of <i>Chd7</i> genotypes amongst 48 genotyped clones. <b>K.</b> Confirmation of <i>Chd7</i> inactivation in individual Chd7^Δ/Δ clones by qRTPCR.</p

Optimization of mESC transfection.

<p><b>A.</b> pCas9-GFP expression plasmid was transfected into mESCs by the indicated method. GFP fluorescence was assessed by fluorescent microscopy and FACS. Numbers indicate the fraction of GFP-expressing cells. <b>B.</b> Percent of transfected cells and mean fluorescence intensity of GFP+ cells. n = 3. Graphs show mean ± s.e.m.</p

Cas9 gene knockout using single or dual gRNAs.

<p><b>A.</b> mESC cell line L10a-GFP, in which one Rosa26 locus expresses GFP. <b>B.</b> Experimental outline and gRNAs. gRNAs #9 and #5 generate 5′ overhangs for dual nickase strategy. <b>C.</b> Single gRNA-directed GFP inactivation. <b>D.</b> Comparison of gRNA alone or gRNA+HDR donor containing a translational stop signal. n = 3. Bar = s.e.m. Red circles indicate individual data points. <b>E.</b> Gene inactivation frequency of single compared to paired gRNAs. <b>F–G.</b> Assessment of GFP expression in mESCs in the GFP-FACS fraction by fluorescent microscopy and western blotting. <b>H.</b> Effective gene knockout using dual gRNAs and the hCas9-D10A nickase mutant.</p