# Computational Tools and Resources Supporting CRISPR-Cas Experiments
## 4 Repair Outcome Predictions
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Compared Feature** & **FORECasT** & **SPROUT** & **inDelphi** \\ \hline \hline \multicolumn{1}{c}{
\begin{tabular}{c} Indel frequency \\ Average indel length \\ Sequences of predictions \\ Alignment of predicted sequences \\ Frequency count for every \\ predicted sequence \\ Frameshift/ \\ in frame frequency \\ MH strength of target site \\ Level of homogeneity \\ of predictions \\ Distinction between MH and \\ MH-less deletions \\ Cell type enable to choose \\ Batch mode \\ Gene mode \\ Shareable link to results \\ Summary statistics \\ for download \\ References & [3] & [82] & [7] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Comparison of repair outcome prediction tools. frequency, this tool presents information on the frameshift frequency and microhomology (MH) strength of a target site. Predicted deletions are described as microhomology-resulted or microhomology-less. Moreover, InDelphi allows choosing the cell type of interest, HEK293, HCT116, K562, mESC and U2 OS, which is required to achieve a high accuracy of prediction. Furthermore, in batch mode, InDelphi allows quick searching for up to 80 gRNAs followed by a chosen PAM in the input sequence and comparing their precision, frameshift frequency and microhomology strength with the results of a single mode for each gRNA. In the gene mode InDelphi scans exonic sequences of selected human or mouse genes to provide hundreds of gRNAs with statistics similar to those in batch mode, and includes information about distances from the 5' and 3' ends of the targeted exon for each gRNA [7]. We compared predictions generated by ForeCast and InDelphi to empirical data obtained in genome editing experiments carried out at two distinct loci. The sites differ in nucleotide sequence architecture: the _TP53_ (tumor protein P53) locus is microhomology-rich and contains homologous tracts in close proximity to the cleavage site (Figure 2A, marked in yellow) making MME) a highly likely mechanism of DNA repair. In the targeted region of _HBB_ (hemoglobin \(\beta\)) longer microhomologies are relatively less abundant. In the case of _TP53_, both algorithms correctly predicted the occurrence of a relatively long, 30 bp deletion as the most probable editing result. Indeed, we observed a biallelic deletion, 30 and 50 bp long at this site in HEK293T cells [47]. The 50 bp deletion was not predicted since it exceeds the indel length capacity of both tools. The results confirm that MMEJ-based repair may be harnessed for highly predictable and precise genome editing. In the case of _HBB_, neither FOREcasT nor InDelphi was able to accurately assess the probability distribution across the range of indels. As reported by [PERSON] et al. introduction of a DSB resulted in an exceptionally high frequency of a one nucleotide insertion at the locus (43.2%) in HEK293T cells, whereas the tools assigned its relatively low probability (3.9% vs. 6.7%, ForeCasT and InDelphi, respectively) [83]. Nonetheless, both tools were able to predict which nucleotide is the most probable to be inserted (cytidine). The principles determining the preference of a nucleotide to be inserted appear to be relatively straightforward--the inserted base is usually homologous to the nucleotide at position \(-4\) from PAM (Figure 2B, marked in blue) [3; 4]. The high frequency of single nucleotide deletions predicted by both algorithms stems from the occurrence of CCCC motif directly at the cutting site. It has been reported that cytosine at the \(-5\) and \(-4\) positions relative to the cutting site results in a high probability of a single nucleotide excision [3; 4]. However, the real frequency of this event turned out to be significantly lower (6.8%) [83].