September 1, 2017 (Vol. 37, No. 15)

DeeAnn Visk Ph.D. Founder and Principal Writer DeeAnn Visk Consulting

Scaling CRISPR Technology to Produce Off-the-Shelf Libraries

The CRISPR/Cas9 system, we are told, is so very easy to implement—and it is, at least initially. But CRISPR/Cas9 may soon run into difficulties. For example, a CRISPR/Cas9-based process may need to be scaled up or streamlined, reoriented toward more sophisticated tasks, or refined to deliver consistent, verifiable results. When that happens, a CRISPR/Cas9 process may betray its improvisational origins or its jerry-rigged nature.

Fortunately, CRISPR/Cas9 is becoming more systematized through the introduction of off-the-shelf tools: oligo libraries, assay kits, selectable markers, and more. These tools, in the hands of creative investigators, enable increasingly powerful genome-engineering platforms—more precise, more efficient, and more capable of delivering modifications that show superior fit and finish, as may be determined with­—you guessed it)—commercially available assays.

A prominent provider of CRISPR/Cas9 tools is GE Healthcare Dharmacon. The company’s products support applications from CRISPR/Cas9 gene editing to functional genomics.

“The entire human genome is available as synthetic CRISPR RNAs (crRNAs) in a 96-well or 384-well plate format, which can easily be used in arrayed phenotypic screens,” states Annaleen Vermeulen, Ph.D., a senior scientist at GE Healthcare Dharmacon. “We design and synthesize crRNAs to target every gene in the genome, and each crRNA is provided in its own individual well.”

The approach permits a phenotypic readout of cell response using an automated microscope. Earlier this year, an example of this approach was described in the Journal of Biotechnology. Essentially, 169 genes pertinent to cell-cycle regulation were targeted for disruption with CRISPR/Cas9 reagents delivered by lipid transfection. Seven different parameters of the cells were measured in a high-content assay.

“One advantage of the system offered by Dharmacon is that we have designed at least four different guide RNAs per gene,” asserts Dr. Vermeulen. “When these different guide RNAs are utilized, the gene to be disrupted is targeted at four different loci. Thus, if the same effect is seen using all four of the guide RNAs for the same gene, then the results are more likely to be biologically relevant and not merely an artifact of the CRISPR/Cas9 editing or an off-target effect.”


Arrayed Library Screening

The arrayed synthetic form of CRISPR/Cas9 allows many different assays (e.g., enzymatic, endpoint, secreted factors) to be performed without resorting to antibiotic selection, long time points, or cell splitting, procedures that may be required with the vector-based single-guide RNA (sgRNA) approach, explains Dr. Vermeulen.

“These additional procedures can cause noise for certain readouts, so expression of sgRNA in arrayed format can be difficult to implement in a high-throughput setting.”

Predesigned or custom synthetic guide RNAs can be ordered on Dharmacon’s website. “Ordering these predesigned reagents greatly simplifies the process of doing a CRISPR/Cas9 experiment,” advises Dr. Vermeulen. “One need not be an expert in the design aspects of CRISPR/Cas9 technology to complete experiments using this technique. Being able to transfect the cells with a lipid reagent or electroporation becomes the only technical requirement.”

The Dharmacom technology supports several experimental approaches. “For example,” notes Dr. Vermeulen, “our system could be used to knockout a gene or genes in a cell line and then assay the cell line with a chemical library of small compounds in a high-throughput screen.”

Compared to RNA interference (RNAi) systems, such as those making use of short hairpin RNA (shRNA), short interfering RNA (siRNA), and double-stranded RNA (dsRNA), CRISPR editing permanently modifies genomic DNA rather than transiently modulating RNA expression levels. The ability to perform permanent gene knockout experiments, in addition to transcription knockdown, provides researchers a more complete understanding of gene function.


Library Generation

Another technical advance is the ability to generate synthetic DNA that has much more integrity than the synthetic DNA that was generated in the past. “We are now able to synthesize DNA with an error rate equal to or better than the current fidelity of next-generation sequencing,” asserts Benjamin Borgo, Ph.D., global senior product manager, Agilent Technologies.

He explains that the company leveraged this increase in DNA fidelity to synthesize ultra-high-quality CRISPR libraries, adding that this enhancement in DNA quality leads to a corresponding improvement in the quality of many of the company’s molecular biology and genomics reagents that rely on synthetic DNA as an input.

“Another exciting advance is the length of DNA we can synthesize, which has increased alongside our improvements in fidelity. People are often amazed to hear that Agilent has the ability to print an entire human genome in a single run on one of our DNA writers,” he says.

The solutions that Agilent has created for library generation of CRISPR oligos provide a complete and general workflow, generating customized plasmid libraries used in high-throughput functional genomics screens, according to Dr. Borgo. End users can now employ a DNA library of their own (or Agilent’s) design, run a simple assembly reaction, and move to a plasmid library in less than two hours of hands-on work, he points out.

Because there are a number of existing solutions, users can introduce errors and biases into the library during characterization, which leads to additional cost and uncertain results when the actual screening is performed, maintains Dr. Borgo.

“The SureGuide libraries developed by Agilent eliminate this quality loss, as well as the associated uncertainty,” he says. “In general, our solution drastically simplifies the most general steps in a pooled CRISPR library workflow and does so in a way that not only maintains the quality of the library, but improves it from step to step.”


Measuring Targeting Efficiency

Other technologies aim to facilitate and accelerate processes that evaluate whether cell lines incorporate CRISPR-induced mutations. Normally, after a CRISPR experiment, scientists must isolate, grow, and analyze several subclones from cell lines to determine whether genetic modifications that had been intended were actually implemented.

“The technology of our process is a basic T7 endonuclease I assay,” says Kyle Luttgeharm, Ph.D., application scientist, Advanced Analytical Technologies (AATI). “AATI has carefully optimized this assay to ensure that the cleavages produced are actually matching the probability of what should be happening in that test tube. We recently published a paper on all of our work in the journal BioTechniques.”

The T7 endonuclease I recognizes mismatches in DNA heteroduplexes by detecting the “bubble” that forms in the double-stranded DNA. “AATI has developed a kit using a highly active T7 endonuclease, AccuCleave™ T7CE,” notes Dr. Luttgeharm. “In combination with our instrument, the Fragment Analyzer Automated CE System, and another kit (CRISPR Discovery Gel Kit), the AccuCleave endonuclease facilitates a high-throughput ability to rapidly screen for clones likely to contain the desired mutations.”

In a traditional research setting, the steps to determine the mutational status of clonal lines after the CRISPR experiment would be to extract genomic DNA, PCR amplify the fragment of interest, and then sequence these fragments. Following these steps for many clones is a laborious, protracted, and expensive proposition. Using the methods and instrumentation from AATI can expedite the process, reducing the amount of resources employed to find the correctly modified cell line.

“Some of our customers in the pharmaceutical industry use this process to screen for modified cell lines,” informs Dr. Luttegeharm. “AATI happily works with our customers to design a workflow that minimizes expenses of these processes. We also work with companies that develop CRISPR applications themselves, to assist them in evaluating the efficacy of their products.”


Deriving an edited monoclonal cell line: Typical work flow with CASPR editing. [Advanced Analytical (AATI)]

The Use of T7 Endonuclease to Expedite Screening

The machinery of CRISPR/Cas9 was discovered in bacteria and archaea, microorganisms in which CRISPR/Cas9 serves as an adaptive immune system. This system, which protects against foreign genetic material, has the previously mentioned component, T7 endonuclease I, which recognizes and cuts double-stranded DNA where there are two or more mismatched DNA base pairs. Efforts to engineer deletions or insertions in a DNA site by CRISPR/Cas9 experiments often result in two or more mismatched base pairs.

The cutting action of T7 endonuclease I can be exploited experimentally, for example, to assess the targeting efficiency of CRISPR/Cas9. Cells that have been subjected to genome editing are lysed to permit the extraction of genomic DNA, which is amplified (for the region of interest) with PCR. After PCR amplification, the DNA undergoes denaturing and reannealing, yielding various reannealing products, some of which constitute heteroduplexes, which are digested with T7 endonuclease I. Then, the undigested DNA and the digestion fragments should be analyzed to generate estimates of targeting efficiency.

These steps, which come from an application note prepared by scientists based at PerkinElmer and other labs, culminate in “visual inspection through digitally represented electropherograms and tabular quantitative data,” which is enabled by the LabChip Gel Xpress (GX) Touch, a PerkinElmer instrument, notes a Perkin Elmer official.

“By collaborating with another company, we produced a workflow that allows automation to speed up detection of changes of interest in the DNA of cells which have undergone CRISPR/Cas9 manipulation,” states Zhiyeng Peng, Ph.D., application scientist, PerkinElmer. “Our new instrument completes the analysis of one sample in about one minute. Traditional microcapillary techniques take much longer.”

“Once the genomic DNA is extracted from the sample of interest, the region of interest in DNA is PCR-amplified and then allowed to slowly reanneal to permit formation of heteroduplex strands of DNA. The amplified DNA is then digested with the T7 endonuclease I. The cleavage only takes place at the heteroduplexes, where there are two or more mismatched base pairs.

“The longer fragments of the PCR amplification are the intact DNAs that have not been cleaved by the highly selective T7 DNAse,” continues Dr. Peng. “By analyzing the results, the quality and quantity of the CRISPR/Cas9 reaction can be evaluated.”


Incorporation of Selection Markers

The challenge of ensuring that both copies of the DNA in a diploid cell line have the mutations of interest can be addressed through the use of selection markers. “We have done the hard work of supplying genome-wide knockout kits,” details Xuan Liu, Ph.D., head of marketing, OriGene. “We have a kit for every single gene in the human and mouse genomes. The donor vectors, designed to supply donor DNA for the CRISPR experiment, also contain selection markers.”

If a selection marker is used, such as an antibiotic-resistance gene, cells that likely carry a CRISPR/Cas-9-induced modification can be isolated. For example, a puromycin resistance gene can be incorporated into a donor vector. Then, after transfection, cells can be challenged with puromycin. The cells that survive are more likely to have been modified as intended.

A donor vector can also be engineered to contain promoter-less green fluorescent protein (GFP) markers. Such markers can be used to evaluate the promoter strength of the replaced gene in the cells for an event that incorporates the marker.

“One potential workflow involves loxP sites, which can be used to flox out both the GFP and puromycin markers while leaving behind the mutated DNA,” suggests Dr. Liu. “Advantageously, the same materials can then be used to do another CRISPR experiment to interrupt the gene of interest on the other allele. Thus, another allele on the second copy of the DNA can be disrupted with the same materials as the first copy.

“Additionally, the GFP can provide a visual marker for cells that have incorporated the DNA of the plasmid vector into its genome. The GFP is an added benefit. It does not have its own promoter; therefore, the GFP is not expressed unless it inserts into a region where a different promoter can be used to drive the expression of the GFP.”

According to Dr. Liu another benefit of OriGene’s kit is the addition of predesigned genome-wide coverage.

“OriGene has done all the hard work of designing both the guide RNAs and corresponding donor vector, assembling them into a kit with a simple protocol. The researchers do not have to become experts on CRISPR to use it in their research,” says Dr. Liu. “Following our step-by-step instruction, a person with zero knowledge of CRISPR can utilize this system or kit.”  


Genetically Modified Mice

CRISPR technology has revolutionized the process for creating genetically modified mice, allowing for shorter timelines and, in many cases, more efficient production.

In a presentation entitled, “Utilizing CRISPR Technology to Develop Mouse Models of Human Disease,” at the Hanson Wade CRISPR 2017 Conference, David S. Grass, Ph.D., senior director of genetic engineering, genotyping, and reproductive sciences at The Jackson Laboratory (JAX), discussed the impact of CRISPR technology on the JAX Model Generation platform. 

Dr. Grass highlighted the ability and experience of the JAX team to create genetic modifications on a wide array of genetic backgrounds (important for creating changes on strains that have preclinical relevance) and contrasted the CRISPR technology with more traditional techniques, such as homologous recombination in mouse embryonic stem (mES) cells, for creating genetic modifications. 

“CRISPR technology increases the efficiency of homologous recombination such that the genetic modification can be performed in single-cell embryos, as opposed to mES cells,” said Dr. Grass. “In addition to decreasing the timelines and the cost for creating the genetically modified mice, this allows for the work to be performed directly on different genetic backgrounds for which robust mES cells don’t exist, avoiding extensive backcrossing from the strain the mutation was made on to the preferred background strain.”

Case studies were presented for two projects performed for Cat Lutz, Ph.D., an investigator at JAX.  For one, an allelic series was created for familial amyotrophic lateral sclerosis (FALS) using an oligo-mediated knockin strategy to create different SNPs in the endogenous Tuba4 gene that are similar to those associated with patients with FALS. 

The other project involved the creation of a model for Friedreich’s Ataxia, for which a conditional Frataxin null allele was created using a double-stranded DNA plasmid-mediated knockin strategy.  The detailed design strategies for both projects were reviewed during the meeting.

The processes for validating the mice also were discussed. These include PCR and sequence analysis,designed to ensure that the desired mutation exists in the genome and has occurred at the targeted locus. 

























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