Designing custom CRISPR libraries for hypothesis-driven drug target discovery

Abstract

Over the last decade Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has been developed into a potent molecular biology tool used to rapidly modify genes or their expression in a multitude of ways. In parallel, CRISPR-based screening approaches have been developed as powerful discovery platforms for dissecting the genetic basis of cellular behavior, as well as for drug target discovery. CRISPR screens can be designed in numerous ways. Here, we give a brief background to CRISPR screens and discuss the pros and cons of different design approaches, including unbiased genome-wide screens that target all known genes, as well as hypothesis-driven custom screens in which selected subsets of genes are targeted ( Fig. 1 ). We provide several suggestions for how a custom screen can be designed, which could broadly serve as inspiration for any experiment that includes candidate gene selection. Finally, we discuss how results from CRISPR screens could be translated into drug development, as well as future trends we foresee in the rapidly evolving CRISPR screen field.

A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system

“…sgRNAs with stabilizing 2′-O-methyl and phosphorothioate linkages were ordered from Sigma-Aldrich or Synthego. The geneMANIA plugin for Cytoscape [24] was used to identify potential interaction partners of HOXB8, as discussed in [25] . Primers were designed using Primer-BLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ), aiming for a 400–800 bp amplicon with the sgRNA binding site in the middle.…”

CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies

“…Hox or B16 cells were spin-infected and subsequently selected using Puromycin or Blasticidin to remove the non-infected cells. sgRNA design, electroporation, and transfection sgRNAs were designed using the Green Listed software (28,29) utilizing sgRNA design from the Doench mouse library (14). 2'-O-methyl and phosphorothioate stabilized sgRNAs (Supplementary Table S2) were ordered from Sigma-Aldrich or Synthego.…”

A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system

“…The Green Listed software (http://greenlisted.cmm.ki.se) [25,26] utilizing the Brie reference library, typically selecting the sgRNA with the highest on-target activity [27], or https://design.synthego.com/#/ were used to design sgRNAs. sgRNAs with stabilizing 2'-Omethyl and phosphorothioate linkages were ordered from Sigma-Aldrich or Synthego.…”

“…sgRNAs with stabilizing 2'-Omethyl and phosphorothioate linkages were ordered from Sigma-Aldrich or Synthego. The geneMANIA plugin for Cytoscape [28] was used to identify potential interaction partners of HOXB8, as discussed in [26]. Primers were designed using Primer-BLAST Shen Y et al, 2021 -5/19 -(https://www.ncbi.nlm.nih.gov/tools/primer-blast/), aiming for a 400-800 bp amplicon with the sgRNA binding site in the middle.…”