SEP 29, 2015 8:00 AM PDT

Improve CRISPR-Cas9 experiments with rationally designed guide RNAs

Sponsored by: Dharmacon, Dharmacon
Speaker

Abstract

To learn more about Gene Editing watch “Webinar: CRISPR-Cas9 Gene Editing with Synthetic RNA – from start to finish!”

 Attendees will learn:

  • the importance of a thorough and accurate guide RNA specificity alignment
  • how rational design of guide RNAs can improve CRISPR functionality
  • the impact of genome-wide, algorithm-designed guide RNAs on gene editing efficacy and time to results

More about the webinar:
The CRISPR-Cas9 system introduces double-strand DNA breaks at a specific locus in the genome by using a complex of the Cas9 nuclease with either a chimeric single guide RNA (sgRNA) or two short RNAs (a CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA)). The ability of any given sgRNA or crRNA to create a break in the target DNA that causes functional protein disruption can vary based on the guide RNA (gRNA) sequence and position in the targeted gene. Likewise, the overall specificity of RNA-directed DNA cleavage events is not yet completely understood and can hamper its wider application.
While gRNAs targeting one or a few genes can often be chosen ad hoc, performing high throughput genome-scale loss-of-function screens requires gRNAs that have consistently high functional knockout efficiencies. To understand the parameters affecting CRISPR-Cas9 gene editing efficiency, we systematically evaluated over 1100 synthetic crRNAs in a reporter assay that directly measures functional activity of a central cellular process (the ubiquitin-proteasome pathway) and identified characteristics important for functional gene disruption. Using this data, we developed and trained an algorithm to score gRNA sequences based on how likely they are to produce functional knockout of targeted genes. We further tested our algorithm by designing synthetic crRNAs to genes unrelated to the proteasome and examined their ability to knock out gene function using additional phenotypic assays, as well as their cleavage efficiency using next-generation sequencing analysis. Our results demonstrate that high-scoring crRNAs have increased functionality. Further, we developed an optimized alignment program to perform complete specificity analysis of crRNAs, including detection of gapped alignments. Recent work has demonstrated gene editing by crRNAs containing bulges of up to 4 nucleotides, but existing design tools are unable to detect putative off-targets based on gapped alignments.  We have combined this comprehensive specificity check with our functionality algorithm to select and score highly specific and functional gRNAs for any given gene target.


Show Resources
You May Also Like
MAR 23, 2022 11:00 AM PDT
MAR 23, 2022 11:00 AM PDT
Date: March 23, 2021 Time: 11:00am (PDT), 2:00pm (EDT), 8:00pm (CEDT) In this presentation, Dr. Middleton will review the development and deployment of large-scale saliva-based COVID-19 test...
JUN 28, 2022 7:00 AM PDT
JUN 28, 2022 7:00 AM PDT
Date: June 28, 2022 Time: 3:00pm (BST), 4:00pm (CET), 9:00am (CST), 7am (PST) Light-sheet microscopy is an extremely versatile imaging technique with a vast range of implementations that are...
APR 26, 2022 7:00 AM PDT
C.E. CREDITS
APR 26, 2022 7:00 AM PDT
Date: April 19, 2022 Time: 7:00am (PDT), 10:00am (EDT), 4:00pm (CEST) High-content (HC) phenotypic profiling approaches are a powerful tool to study the effect of biological, genetic, and ch...
MAY 17, 2022 9:00 AM PDT
MAY 17, 2022 9:00 AM PDT
Date: May 17, 2022 Time: 9:00am (PDT), 12:00pm (EDT), 8:00pm (CEST) Gene therapeutics have great potential to treat many severe diseases in an unprecedented, targeted manner. The biopharmace...
JUN 21, 2022 6:00 AM PDT
JUN 21, 2022 6:00 AM PDT
Date: June 21, 2022 Time: 6:00am (PDT), 9:00am (EDT), 3:00pm (CEST) The global understanding and practice of medicine is currently undergoing a revolutionary change. This shift to precision...
MAR 02, 2022 9:00 AM PST
C.E. CREDITS
MAR 02, 2022 9:00 AM PST
Date: March 02, 2022 Time: 9:00am (PST), 12:00pm (EST) Single cell RNA-seq is known to only capture a small fraction of the transcriptome of each cell. Often, this is due to inherent limitat...
Loading Comments...
Show Resources