Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion

Abstract

CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.

Enhancing CRISPR deletion via pharmacological delay of DNA-PK

“…We observed wide variability in the deletion efficiency of sgRNA pairs, from Pair1 (P1) displaying minimal efficacy, to the most efficient P4 yielding ~40% deletion. Therefore these paired sgRNAs achieve deletion efficiencies that are comparable to previous studies 4,26 . Measured deletion rates were consistent across biological replicates (Fig.…”

“…The principal drawback of CRISPR-del is the low efficiency with which targeted alleles are deleted. Studies on cultured cells typically report efficiencies in the range 0% -50% of alleles, and often <20% 29,30 , similar to estimates from individual clones 4,17,[24][25][26] . Indeed, a recent publication reported high variation in the efficiencies of paired sgRNA targeting the same region, including many that yielded negligible deletion 30 .…”

“…We next assessed whether these effects hold for other loci and readouts. We previously used a quantitative PCR method (quantitative CRISPR PCR, QC-PCR) to measure rates of deletion at the MALAT1 enhancer region 26 . For three out of four sgRNA pairs, we observed a significant enhancement of deletion with M3814 treatment of HeLa (Fig.…”

“…Thereafter it relies on the endogenous non-homologous end joining (NHEJ) process to repair the breaks so as to eject the intervening fragment [22][23][24][25] . The two ends of target regions are defined by a pair of user-designed sgRNAs 26 . Paired sgRNAs may be delivered by transfection or viral transduction 17,25 .…”

Transcriptome Analysis of Non‐Coding RNAs in Livestock Species: Elucidating the Ambiguity

“…Equally, bioinformatics designs for CRISPR deletions are now possible with a tool known as CRISPETa developed with eicient CRISPR deletion of an enhancer and exonic fragment of MALAT1, a lncRNA. CRISPETa can be used for single target regions or thousands of targets and has high-coverage library designs for entire classes of non-coding elements which can be adopted for use in livestock species [226]. CRISPR-Cas9 may be used with a gene drive incorporated with genome edit to investigate the control of any biological process and can be used to accelerate livestock breeding [225].…”