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High‐throughput analysis of CRISPR RNA seed sequence identity
Author(s) -
Phan Phong T,
Sashital Dipali
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.626.4
Subject(s) - trans activating crrna , crispr , biology , genetics , rna , dna , effector , computational biology , crispr interference , cas9 , gene , microbiology and biotechnology
The type I‐E CRISPR–Cas (clustered regularly interspaced short palindromic repeat–CRISPR associated) system allows bacteria to fight off bacteriophage and other invader threats. During CRISPR interference, CRISPR‐derived RNAs (crRNAs) guide Cas effector proteins to base pair with complementary sequences in the foreign dsDNA, which leads to the cleavage of the DNA target. Target binding occurs directionally from the 5′‐end of the crRNA guide, and the first several base pairs formed between the crRNA and DNA are important to ensure binding. Mutation within this “seed” sequence often reduces the level of CRISPR interference and promotes an alternative “priming” mechanism. During priming, segments of the foreign DNA are inserted into the CRISPR array to create templates for new crRNAs. Our previous results have shown that for some crRNA sequences, mutations within the seed region can be tolerated. Here, we systematically tested the effects of the crRNA seed sequences on CRISPR interference and priming activities. Using partially randomized seed libraries, we identified the position and nucleotide‐specific effects on the mismatch tolerance for four Escherichia coli strains expressing crRNAs with different seed sequences. We show that G/C‐rich seed sequences are more tolerant of mutations than A/U‐rich seed sequences. We observe that guanine mismatches cause the strongest CRISPR immunity defects for all seed sequences tested. G/C‐rich crRNAs may promote CRISPR immunity against mutated sequences at higher rates due to the lower probability of encountering guanine mismatches. Our results indicate that crRNA sequences with higher G/C content may provide stronger immunity than A/U‐rich crRNAs. Support or Funding Information R01 GM115874 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .