# Computational Tools and Resources Supporting CRISPR-Cas Experiments
## 2 Experimental Design--the Choice of a Genome Editing System  
Naturally, the research goals determine the model of choice, the targeted genomic site and the course of the experiment. There are, however, considerations particularly important in the context of CRISPR-Cas research. The crucial decisions concern the choice of Cas nuclease, format of CRISPR-Cas components and delivery of the system to the cell. The choices are particularly determined by the aim of the experiment (knock, knockout, base editing, need for transient or stable nuclease activity), as well as the model used and its susceptibility to different forms of transfection or transduction. SpCas9 is the most widely used nuclease in genome engineering, but advancements in the identification of its orthologs have resulted in a repertoire of nucleases characterized by different sizes, PAM requirements and cleavage patterns (e.g., Cas9s of _Staphylococcus aureus_ and _Streptococcus thermophilus_ or Cas12a of _Francisella novicida_) [32]. The other branch of research has resulted in \"rationally engineered\" nucleases with even more improved specificity [33]. The other strategies that proved useful in certain applications are the use of nickase (Cas9n) and catalytically inactive version of Cas9 (dCas9) [24, 25, 34, 35]. Most of the gRNA design tools integrate PAM requirements and cleavage profile of many nucleases offering a considerable degree of flexibility in aiming at the desired editing site. The format of CRISPR-Cas components (DNA, mRNA, RNP complex) may impact the efficiency and specificity of the nuclease. Purified Cas9 protein and in vitro transcribed gRNA can be combined to form the RNP complex delivered subsequently by electroporation or lipofection. This format leads to immediate yet short-lasting nuclease activity, as opposed to a more stable and longer expression from  
Figure 1: **(A)**. The overview of a CRISPR-Cas experiment. Preliminary analysis involves screening of gRNA activity. Results analysis refers to NGS data analysis. **(B)**. The gRNA design step involves the prediction of the gRNA efficiency (on-target activity) at a given _locus_ and specificity (off-target activity); **(C)**. The most probable results of CRISPR-Cas edits can be predicted by the analysis of microhomologies and by exploiting the known biases in the distribution of repair outcomes. **(D)**. Web-based tools facilitating the post-experimental sequencing data analysis. MH—microhomology. a plasmid. The peak of Cas9 activity delivered in the form of RNP complex occurs a few hours after transfection and then declines quickly as gRNA and Cas9 protein are degraded, leading to a significant increase in specificity [36; 37]. A strategy based on a similar rationale utilizes in vitro transcribed Cas9 mRNA and gRNA [38; 39]. Lipofection or calcium phosphate transfection are high-throughput and cost-effective delivery methods but may be not suitable in the case of difficult to transfect cells, such as primary or stem cells. Electroporation or viral transduction may be used instead but are more demanding in terms of labor and expense. Viral, specifically lentiviral, delivery allows a stable integration of genes into the genome. Adeno-associated viruses (AAVs) are preferable due to the relatively low immunogenicity but their packaging limit renders simultaneous delivery of sgRNA and SpCas9 gene problematic [40; 41]. Another factor worth considering is that different cells exhibit a bias towards different DNA repair outcomes [3; 6]. The phenomenon may be potentially levered to increase the chance of obtaining the desired type of edits. Services such as Addgene or IDT offer not only plasmids, proteins and oligos necessary for CRISPR-Cas experiments but also repositories of protocols that the researcher may find useful at the experiment planning stage [42; 43]. Springer Nature provides a browser of CRISPR-related methods papers, whereas Bio-protocol has released a curated collection of CRISPR-Cas-related protocols [44; 45].