A recent paper by the Lee et al (2018) has shown that the present CRISPR/Cas9 design tools do not accurately predict the activity of single-gene targeting gRNA sequences. They also report that the non sequence based features like DNA accessibility influence the Cas9 activity and suggests incorporation of locus specific metrics in the algorithms of next generation gRNA design tools.
The CRISPR/Cas9 system (Clustered regulatory interspaced short palindromic repeats /CRISPR-associated protein 9) is adopted from Streptococcus pyogenes (Spy) and is a adaptive immune system present in bacteria and archaea that confers resistance to foreign DNA. It consist of two components i.e. guide RNA (gRNA) and Cas9 system. The gRNA is a short single stranded RNA sequence with an 80-nucleotide constant region and a short 20 nucleotide target specific sequence that binds to the target DNA. By altering the short 20-nucleotide sequence, it is possible to target almost any 20bp sequence in the human genome. However, different gRNAs have varying degree of activity, with some gRNA capable of targeting 21% of alleles in a population of cells , whereas, other achieved targeting rates of 77% ( Cradick et al 2013 ; Lee al 2017). This inconsistency has led to the development of several CRISPR/Cas9 tools for the search of highly active gRNA design.
There are several publically available design tools like sgRNA designer, CRISPRScan etc. However, many of these do not directly measure gRNA activity. For example, some studies determine gRNA efficiency by the loss of a surface marker, thereby counting only those cells that had undergone biallelic frameshift mutations (Doench et al 2014).
In the present study Lee et al demonstrated that the existing publicly available design tools have low predictive power to predict the efficiency of gRNAs for single- gene- targeting. They tested 198 gRNAs individually and compared their score with the observed level of activity and found that the best performing algorithm had an R2 of 0.3, suggesting poor predictive capability.
Apart from these design algorithms and sequence-based features like secondary structure or folding properties, non sequence based features like DNA accessibility and chromatin state also influence Cas9 activity. Recent in vitro studies have shown that DNA packed as nucleosomes are protected from Cas9 cleavage.
Lee et al studied the effect on DNA accessibility in mammalian cells by designing a series of gRNAs that target repetitive elements .These gRNA have 4- 20,000 target sites in human genome. Individual target sites were isolated by barcoded primers unique for each locus. Thus, limiting the sites to be analyzed. The level of CRISPR /Cas9 activity varied significantly across all sites, suggesting that Cas9 activity is influences by locus specific feature in spite of the target sequence.
This report demonstrated that the predictive power of first generation CRISPRgRNA design tools do not accurately predict the cleavage efficiency of Cas9 for single–locus gene modifications in human cells and also suggests that non sequence-based features like DNA accessibility influence Cas9 activity. It further suggests that the predictive power of new generation gRNA design tools can be increased by including both sequence and target site accessibility metrics.
Cradick, T. J., Fine, E. J., Antico, C. J., & Bao, G. (2013). CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Research, 41(20), 9584–9592. http://doi.org/10.1093/nar/gkt714
Doench, J. G., Hartenian, E., Graham, D. B., Tothova, Z., Hegde, M., Smith, I., … Root, D. E. (2014). Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nature Biotechnology, 32(12), 1262–1267. http://doi.org/10.1038/nbt.3026