IN SILICO ANALYSIS OF POTENTIAL OFF-TARGET SITES TO GENE EDITING FOR MUCOPOLYSACCHARIDOSIS TYPE I BY USING THE CRISPR/CAS9 SYSTEM: IMPLICATIONS FOR POPULATIONS-SPECIFIC TREATMENTS

Background The CRISPR/Cas9 system is an accessible and less onerous editing system to treat human genetic diseases, but off-target prediction is paramount to ensure the safety and efficiency of this system in clinical applications. The aim of this study is to evaluate off-target regions for a guide RNA (sgRNA) designed to correct the most common variant found in Mucopolysaccharidosis type I (MPS I) patients and also possible implications for population-specific treatment to treat genetic diseases. Methods The most employed in silico tools for off-targets prediction were used: CHOPCHOP, COSMID, Cas-OFFinder, CCTop and CRISPOR. Moreover, new PAM sequences (NNG or NGN) were also evaluated by using Burrows-Wheeler Aligner for this sgRNA against human genome version hg19. The potential off-target regions were selected from sequences selected by the tools’ score and with up to 6 mismatches and up to 2 indels, and the conservation of 5 nucleotides upstream PAM (SEED). The polymorphic site evaluation was made using 1000 Genomes and Latin America databases (ABraOM and LOVD 3-Argentina). Polymorphic sites that have equal or greater 1% in at least one of the populations were selected. Results From a total of 13,422 off-targets, 272 potential off-targets were selected for this sgRNA. A total of 84 polymorphic sites were recognized in the off-target sequences. The 1000 Genomes had the highest amount of polymorphic sites, whereas ABraOM reported only six. In most cases polymorphic sites decrease the probability of off-target cleavage by increasing the number of mismatches and/or indels and also disrupt PAM and SEED regions. On the other hand, one off-target sequence not previously recognized by all the five predictors was created by a polymorphic variant with frequencies of 42% in African populations and of 16% in European and Latin American populations. Conclusion Here we show the importance of identifying potential off-target sites considering the analysis of frequent polymorphic sites in different populations. Thus, this analysis contributes to a better evaluation of safety aspects of gene editing to treat MPS I patients and other genetic diseases in a population-specific context by using the CRISPR/Cas9 system.