CRISPR Gene Editing

The gene/genome editing landscape changed in 2012 and 2013 when a series of papers described the use of CRISPR as an efficient, and comparatively easy, method for gene editing.

AATI CRISPR Photo

What is CRISPR?

CRISPR/Cas9 genome editing was derived from the naturally occurring defense mechanism used by bacteria to shield themselves from infection by bacteriophages and mobile genetic elements. In nature, CRISPR functions as a bacterial adaptive immune system, releasing RNAs that are complementary to intruding DNA – marking them for destruction. Encounters with bacteriophages and mobile genetic elements are stored in the bacterial genome in spacer segments sandwiched between short, repeating palindromic sequences of DNA. These repetitions and spacer DNA build-up over time, tracking bacterial encounters with hostile nucleic acids through millions of years.

Scientists were able to use the biology of the CRISPR adaptive immune system by altering guide RNAs and transfecting a CRISPR-associated nuclease, Cas9. Due to its simplicity and low cost, CRISPR/Cas9 technology was rapidly adopted by a diverse range of laboratories.

Guide RNAs – single guide RNAs (sgRNA) are commonly used – are short synthetic oligonucleotide sequences that are complementary to a targeted DNA sequence. The sgRNA is part of a longer RNA molecule that forms a riboprotein with the Cas9 enzyme machinery. The guide RNA positions the Cas9 enzyme to the correct position on the target DNA for cleavage. The result is a programmable nuclease that can efficiently target any sequence within nearly any genome of interest.

CRISPR editing technology offers significant advantages over preceding genome editing technology, such as Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). With a lower cost, shorter learning curve, increased efficiency, and the ability to target multiple genes simultaneously, CRISPR technology outstrips other gene editing technologies.

CRISPR genome editing has changed how much of molecular biology research is done on a fundamental level. With a low cost, a low learning curve, and a high degree of accuracy, CRISPR technology has been adopted much more rapidly than ZFNs and TALENs. To optimize CRISPR workflow efficiency, quality reagents must be used, a proper screening technique must be employed, and positive clones must be characterized accurately. Of these three, characterization of positive clones receives the most attention, while screening methods and reagent QC are often overlooked. Characterization of CRISPR modified lines requires Sanger sequencing or next-generation sequencing (NGS). While essential to genome editing workflows, sequencing is not suitable as a screening protocol due to the cost, resource requirements, and time.

Numerous methods of screening for positive clones in CRISPR workflows have been developed, including:

  • Heteroduplex cleavage assays
  • High-resolution melt curves
  • qPCR
  • Heteroduplex mobility assays

As screening methods, most of these require specialized knowledge, reducing their accessibility to researchers at large. Furthermore, they are usually unable to determine the zygosity of the introduced modification(s), require subjective data analysis (in the case of high-resolution melt curves), and do not always scale well to the throughput demands of researchers. Combined, these factors make traditional methods of screening of limited efficacy in CRISPR workflows.

CRISPR technology is being used in a variety of applications, many with medical, industrial, and food security implications. Notably, CRISPR has potential roles in developing novel treatments for cancer. Furthermore, CRISPR technology has moved beyond only being a tool for genome editing.

Advanced Analytical Technologies, Inc. has developed optimized screening protocols and QC analysis solutions for CRISPR workflows. An optimized heteroduplex cleavage assay (Download Biotechniques Report: "Heteroduplex cleavage assay for screening of probable zygosities resulting from CRISPR mutations in diploid single cell lines") uses T7 Endonuclease I to address the challenges associated with other screening protocols including zygosity determination and scalability. Similarly, understanding the quality of CRISPR reagents to be used in a genome editing workflow – namely single guide RNA (sgRNA) and gauging polyadenylation of Cas9 mRNA – is of paramount importance to success.

Screening for Positive Clones

The screening solutions from Advanced Analytical Technologies, Inc. (AATI) enable the successful identification and characterization of CRISPR induced modifications introduced by both the Non-Homologous End-Joining and the Homology Directed Repair pathways (Download Application Note: "Use of an Engineered Restriction Site to Estimate CRISPR Homology Directed Repair Efficiency"). Dependent on T7 Endonuclease I, the DNF-440 AccuCleave T7CE Kit is a heteroduplex cleavage assay specially designed by scientists at AATI for use with the DNF-910CP CRISPR Discovery Gel Kit on the Fragment Analyzer™ Automated CE System or the Fragment Analyzer INFINITY® Automated CE System. The CRISPR Plugins (developed in conjunction with Integrated DNA Technologies, Inc.) for PROSize® Data Analysis Software enables researchers to both quickly identify positive clones and determine the zygosity of successfully modified diploid organisms/cells. In diploid organisms, zygosity can take one of three configurations:

  1. Diallelic homozygous: identical modifications on both alleles
  2. Diallelic heterozygous: unique modifications on both alleles
  3. Monoallelic: modification on one allele

By identifying both positive clones and determining zygosity, researchers are given further selection criteria. Now researchers can further characterize only those clones that match more stringent criteria.

While the Fragment Analyzer and the Fragment Analyzer INFINITY can separate up to 96 CRISPR samples in parallel and – in the case of the Fragment Analyzer INFINITY - screen over 2,400 samples per day, sometimes a small-scale approach is warranted. Fortunately, AATI developed the DNA-100 AccuCleave T7 Kit for users who want a low-throughput screening option. Using a variant of the rigorously designed heteroduplex cleavage assay found in the DNF-440 AccuCleave T7CE Kit, this Kit is optimized for detection on agarose gels.

Figure 1. Example separation of a positive clone with diallelic homozygous zygosity. Heteroduplex cleavage assay performed using the DNF-440 AccuCleave T7CE kit. Capillary electrophoresis carried out using the DNF-910CP CRISPR Discovery Gel Kit on a Fragment Analyzer Automated CE System equipped with a Short Capillary Array (33-55).

QC CRISPR Reagents

Figure 2. Quality control analysis of sgRNA. Low amounts of secondary structure and a strong linear peak are desirable for optimal sgRNA performance. Capillary electrophoresis was performed using the DNF-470 Small RNA Analysis Kit on a Fragment Analyzer Automated CE System equipped with a Short Capillary Array (33-55).

Success in CRISPR editing workflows – like all other molecular biology applications – requires the use of reagents that meet suitable quality standards. Single guide RNA (sgRNA), the "guidance" component of CRISPR technology is one such reagent. If sgRNA is not pure enough or has partially degraded, the risks include reduced modification efficiencies and potential off-target effects. Using the DNF-470 Small RNA Analysis Kit or the DNF-472 High Sensitivity RNA Analysis Kit with the Fragment Analyzer or Fragment Analyzer INFINITY allows researchers to ascertain quality of sgRNA (Download Application Note: "Single Guide RNA Quality Assessment By Fragment Analyzer") prior to further use.

A common challenge encountered by researchers employing CRISPR technology is the difficulty associated with transfecting the Cas9 protein into the desired cells. As Cas9 is a large protein, it is difficult to transfect into many cells and nearly impossible in others. To circumvent this challenge, many researchers transfect the Cas9 mRNA and rely on the cells translation machinary to produce Cas9. To offer protection and modulate longevity, the Cas9 mRNA is polyadenylated (Poly(A)). The longer the Poly(A) tail, the longer the Cas9 mRNA can be translated. The DNF-472 High Sensitivity RNA Analysis Kit allows the accurate assessment of Cas9 mRNA quality and Poly(A) tail length, as discussed in this Application Note (Download Application Note: "Analysis of In Vitro Transcribed Cas9 mRNA Pre- and Post-Polyadenylation" ).

Figure 3. Electropherogram of Cas9 mRNA after a 50 minute polyadenylation reaction, generating a 6,197 nt transcript for transfection. Capillary electrophoresis performed using the DNF-472 High Sensitivity RNA Analysis Kit on a Fragment Analyzer Automated CE System equipped with a Short Capillary Array (33-55).

CRISPR Gene Detection Analysis Kits

Advanced Analytical Technologies, Inc. (AATI) provides numerous solutions for CRISPR/Cas9 workflows, from reagent QC to screening for CRISPR-induced modifications. QC solutions using the Fragment Analyzer provide users with confident assessment of Cas9 mRNA and sgRNA suitability, saving researchers time and money. Furthermore, the AccuCleave family of kits and the CRISPR Discovery Gel Kit provide researchers with CRISPR mutation screening protocols adaptable to any throughput.

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