How to design gRNA for CRISPR genome editing?

Because most current gRNA design tools were developed from the data of higher eukaryotic cells, they may not accurately predict gRNA activity in Leishmania . Nevertheless, to design a gRNA with no off-target site(s) that is likely to be active in Leishmania, we recommend using the Eukaryotic Pathogen CRISPR guide RNA Design Tool (EuPaGDT) (http://grna.ctegd.uga.edu/) which ranks a gRNA based on its activity score, off-target sites in the genome and microhomology sequences flanking the double-strand break (DSB) site . CRISPRater (https://crispr.cos.uni-heidelberg.de/) developed from human and mouse data could also be helpful as it can predict gRNA activity with only the guide sequence information . For convenience, one may also directly use the gRNA sequences designed for targeting the immediate 5′ and 3′ flanking sequences for each of Leishmania genes from (http://leishgedit.net/).

References:

• Zhang WW, Matlashewski G (2015) Edición del genoma mediada por CRISPR-Cas9 en Leishmania donovani. MBio 6 (4): e00861
  
  
• Zhang WW, Matlashewski G (2019) El recocido de una sola hebra juega un papel importante en la reparación de rotura de ADN de doble hebra tras la escisión CRISPR-Cas9 en Leishmania. mSphere 4: e00408–19. https://doi.org/10.1128/mSphere.00408-19 
  
  
• Peng D, Tarleton R (2015) EuPaGDT: una herramienta web diseñada para diseñar ARN guía CRISPR para patógenos eucariotas. Microb Genom 1 (4): e000033
  
  
• Labuhn M, Adams FF, Ng M, Knoess S, Schambach A, Charpentier EM, Schwarzer A, Mateo JL, Klusmann JH, Heckl D (2018) La predicción refinada de la eficacia del sgRNA mejora las aplicaciones CRISPR-Cas9 a gran y pequeña escala. Ácidos nucleicos Res 46 (3): 1375-1385