Methods and systems for identifying crispr/cas off-target sites

Abstract

Methods and systems for searching genomes for potential CRISPR off-target sites are provided. In preferred embodiments, the methods include identifying possible on- and off-target cleavage sites and / or ranking the potential off-target sites based on the number and location of mismatches, insertions, and / or deletions in the gRNA guide sequence relative to the genomic DNA sequence at a putative target site in the genome. These methods allow for the selection of better target sites and / or experimental confirmation of off-target sites and are an improvement over partial search mechanisms that fail to locate every possible target site.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation application of U.S. patent application Ser. No. 15 / 114,799, filed Jul. 26, 2016, which was a 371 application of International Application No. PCT / US2015 / 013134 filed Jul. 27, 2015, which claims the benefit of and priority to U.S. Ser. No. 61 / 932,003 filed Jan. 27, 2014 and which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH[0002]This invention was made with government support under Grant PN2EY018244 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING[0003]The Sequence Listing submitted on May 13, 2019, as a text file named “GTRC_6478_SL_txt.txt” created on May 13, 2019, and having a size of 271,348 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).FIELD OF THE INVENTION[0004]The invention is generally directed to bioinformatics methods and systems for identifying CRISPR...

AI Technical Summary

Benefits of technology

[0011]Methods and systems for searching genomes for potential CRISPR off-target sites are provided. In preferred embodiments, the methods include ranking the potential off-target sites based on the number and location of mismatches, insertions, and / or deletions in the gRNA guide sequence relative to the genomic DNA sequence at a putative target site in the genome, allowing the selection of better target sites and / or experimental confirmation of off-target sites.

[0017]The report can include a score indicating the likelihood that the guide sequence will direct a CRISPR / Cas system to the DNA sequence and facilitate nuclease cleavage. The score can be used to rank the putative target sites in a list. The score can include additional information from experiments and / or databases, such as ENCODE, about the genomic context. For example, data on the histones, protein binding or confirmation of individual chromosomal regions can indicate if there is less or more likelihood of cleavage. In some embodiments, target cleavage locations including genomic sequences with higher sequence identity to the guide sequence receive a lower score relative to target cleavage locations having genomic sequences with lower sequence identity to the guide sequence. Typically, in such embodiments, increasing numbers of substitutions, deletions, and insertions at the target cleavage location increase the score, as do substitutions, deletions, and / or insertions closer to the PAM. The scoring mechanism and position weights can be changed to alter the scoring to better model certain CRISPR / Cas activities. For example, in some embodiments, the score is increased more for deletion(s) in the genomic sequence relative to the guide sequence (RNA bulges) than for insertions in the genomic sequence relative to the guide sequence (DNA bulges). The score can also reflect that sgRNA bulges are less tolerant to additional base mismatches, and vice versa.

[0023]The genomic sequence(s) can be DNA sequence converted into FASTA or similarly formatted files, then transformed into index entries that have all possible 25 bases-long tags in the DNA sequence. In other embodiments, other tagging schemes can be used including longer and shorter tags. The index entries can be sorted and the results stored as a binary main index file. The main index file can be divided into parts, each representing entries having about 12 nucleotides of the first nucleotides identical. In other embodiments, other lengths of index files may be used. A secondary index file can include the position in the main index file where each part starts added to the end of the index file. Searching genome sequence organized and indexed in such a way can improve the speed of the search, while allowing exhaustive searching. Preferred embodiments utilize index files, though other embodiments could use other index methods, similar expedited search strategies, or provide searching without index files, as done with linear searches through the full sequence space, though these would increase run times. A particular embodiment of the disclosed method is referred to herein as COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions).

[0024]The disclosed methods and systems can aid the design and optimization of CRISPR guide strands by selecting the preferred target sites with minimum Cas-induced off-target cleavage and facilitate the experimental confirmation of off-target activity by providing both putative off-target sites and primer for testing cleavage that the sites in a CRISPR / Cas system. In some embodiments, the disclosed methods are more exhaustive and / or have a higher sensitivity for identifying putative and / or actual off-target sites than previously known methods or programs.

Problems solved by technology

However, none of these bioinformatics search tools has considered the off-target sites due to insertions or deletions between target DNA and guide RNA sequences, nor provide application-specific primers.

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