CRISPR Gene Editing

CRISPR/Cas9, the gene editing tool that enables highly-efficient and precise modifications to DNA, has been a disruptive force in scientific research and drug discovery. While CRISPR possesses significant ease-of-use advantages over former gene editing methods, it is an advanced tool requiring careful experimental design for optimal outcomes.

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Charles River acquired a license from the Broad Institute to work with CRISPR in May 2014. Charles River biologists employ CRISPR for target screening alone or alongside RNAi screening with our shRNA library (SilenceSelect®) and to create complex custom knock-in and knockout cell lines with applications throughout the drug discovery continuum:

  • Target discovery and validation: gRNA library screening, disease-relevant phenotypic screening
  • Hit discovery: Generate knock-in cell lines expressing your gene of interest to use in cell-based HTS assays
  • Hit-to-lead: Precision mutagenesis of tumor cell lines
  • Lead-to-candidate: ES cell modification for custom transgenic mouse models

Below are example project timelines:

CRISPR-Cas9 used under licenses to granted and pending US and international patents from The Broad Institute and ERS Genomics Limited.

The CRISPR system consists of a guide RNA (gRNA), which defines the genomic target and therefore conveys specificity of the system, and a Cas9 protein with endonuclease activity. Upon binding, the Cas9:gRNA complex generates a double-strand break (DSB) in the genomic DNA. This break can be repaired by two different mechanisms: non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone repair pathway known to create indel mutations in the genome, and therefore often employed to generate gene knockouts. For precise genome editing, repair can also take place via the HDR pathway, in which a repair template with homologous arms to the target sequence is introduced alongside the CRISPR system.

Although the generation of gene knockouts via the NHEJ pathway is fairly straightforward, identifying the clone of interest can be tedious and time-consuming as many different indels can be generated in the poorly controlled repair process, including in-frame deletions that can potentially lead to an aberrant but active protein. At Charles River, we therefore also use the high-fidelity HDR pathway to generate gene knockouts. This allows complete control over the process, confidently generating knockouts as designed. In addition, the HDR pathway is used to introduce transgenes in safe harbor loci, generate specific mutations in a gene or perform SNP corrections.

NHEJ efficiency and sequencing data

In this example, NHEJ was employed to generate a knockout in mouse C57Bl/6NCrl ESC. (A) 73% gene efficiency was achieved. (B) However, sequencing identified 3 out of 4 of the resulting indels as small in-frame deletions; the frequency of homozygosity was 14%.

HDR efficiency and sequencing data.jpg

In this example, the homology-directed repair (HDR) pathway uses (A) a single-stranded repair template to correct the double-strand break, including a stop codon, enabling (B) PCR validation prior to sequencing. While gene editing efficiency was lower than observed with NHEJ (C), homozygous frequency was equivalent (C, D).

Our experts work with proven tools to select the appropriate gRNAs for each project, weighing both efficiency and predicted off-target effects in the decision process. We employ a two-vector system to house the Cas9 and gRNAs, which can be delivered by various mechanisms (nucleofection, transfection). Besides the plasmid system, we also provide the tools in adenoviral vectors to be able to perform CRISPR/Cas9 editing in cells that are difficult to transfect. In addition, the gRNA vector is designed for high-throughput cloning to enable the construction of an arrayed gRNA library for screening purposes.

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