Dataset Information

CRIS.py: A Versatile and High-throughput Analysis Program for CRISPR-based Genome Editing.

ABSTRACT: CRISPR-Cas9 technology allows the creation of user-defined genomic modifications in cells and whole organisms. However, quantifying editing rates in pools of cells or identifying correctly edited clones is tedious. Targeted next-generation sequencing provides a high-throughput platform for optimizing editing reagents and identifying correctly modified clones, but the large amount of data produced can be difficult to analyze. Here, we present CRIS.py, a simple and highly versatile python-based program which concurrently analyzes next-generation sequencing data for both knock-out and multiple user-specified knock-in modifications from one or many edited samples. Compared to available NGS analysis programs for CRISPR based-editing, CRIS.py has many advantages: (1) the ability to analyze from one to thousands of samples at once, (2) the capacity to check each sample for multiple sequence modifications, including those induced by base-editors, (3) an output in an easily searchable file format enabling users to quickly sort through and identify correctly targeted clones.

SUBMITTER: Connelly JP

PROVIDER: S-EPMC6414496 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Publications

CRIS.py: A Versatile and High-throughput Analysis Program for CRISPR-based Genome Editing.

Connelly Jon P JP Pruett-Miller Shondra M SM

Scientific reports 20190312 1

CRISPR-Cas9 technology allows the creation of user-defined genomic modifications in cells and whole organisms. However, quantifying editing rates in pools of cells or identifying correctly edited clones is tedious. Targeted next-generation sequencing provides a high-throughput platform for optimizing editing reagents and identifying correctly modified clones, but the large amount of data produced can be difficult to analyze. Here, we present CRIS.py, a simple and highly versatile python-based pr ...[more]

PMID: 30862905

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Project description:With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.

Dataset Information

CRIS.py: A Versatile and High-throughput Analysis Program for CRISPR-based Genome Editing.

Publications

CRIS.py: A Versatile and High-throughput Analysis Program for CRISPR-based Genome Editing.

Similar Datasets

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