Dataset Information

CRISPR/Cas System and Factors Affecting Its Precision and Efficiency.

ABSTRACT: The diverse applications of genetically modified cells and organisms require more precise and efficient genome-editing tool such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas). The CRISPR/Cas system was originally discovered in bacteria as a part of adaptive-immune system with multiple types. Its engineered versions involve multiple host DNA-repair pathways in order to perform genome editing in host cells. However, it is still challenging to get maximum genome-editing efficiency with fewer or no off-targets. Here, we focused on factors affecting the genome-editing efficiency and precision of CRISPR/Cas system along with its defense-mechanism, orthologues, and applications.

SUBMITTER: Javaid N

PROVIDER: S-EPMC8652214 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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CRISPR/Cas System and Factors Affecting Its Precision and Efficiency.

Javaid Nasir N Choi Sangdun S

Frontiers in cell and developmental biology 20211124

The diverse applications of genetically modified cells and organisms require more precise and efficient genome-editing tool such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas). The CRISPR/Cas system was originally discovered in bacteria as a part of adaptive-immune system with multiple types. Its engineered versions involve multiple host DNA-repair pathways in order to perform genome editing in host cells. However, it is still challenging to g ...[more]

PMID: 34901007

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Project description:BACKGROUND:The presence and activity of CRISPR-Cas defense systems is a hallmark of many prokaryotic microorganisms. Here, the distribution of sequences related to the highly iterated palindrome 1 (HIP1) element and the DNA methylation of CGATCG motifs embedded within HIP1 as a vital part of the CRISPR1 repeat sequence was analyzed in the cyanobacterium Synechocystis sp. PCC 6803. Previously suggested functions of HIP1 include organization of chromosomal structure, DNA recombination or gene regulation, all of which could be relevant in CRISPR-Cas functionality. RESULTS:The CRISPR1 repeat-spacer array contains more than 50 CGATCG elements that are double-methylated (5mCG6mATCG) by the enzymes M.Ssp6803I and M.Ssp6803III. Hence, more than 200 possible methylation events cluster over a stretch of 3600 bp of double-stranded DNA. Bisulfite sequencing showed that these motifs were highly methylated at the m5CGATCG positions whereas specific motifs within the CRISPR1 cas genes were hypomethylated suggesting a lowered accessibility for the DNA methylase to these regions. Assays for conjugation and CRISPR1-mediated DNA interference revealed a 50% drop in conjugation efficiency in the mutant lacking the 5mC methylation of CGATCG motifs, while the highly efficient DNA interference activity was not affected by the lack of m5CGATCG DNA-methylation, nor was the capability to differentiate between self and non-self targets based on the protospacer adjacent motifs (PAMs) GTA and GTC versus the non-PAM AGC. A third DNA methylation mediated by M.Ssp6803II modifies the first cytosine in the motif GGCC yielding GGm4CC. We found a remarkable absence of GGCC motifs and hence the corresponding methylation over an 11 kb stretch encompassing all the cas genes involved in interference and crRNA maturation but not adaptation of the CRISPR1 system. CONCLUSIONS:The lack of GGCC tetranucleotides along the CRISPR1 interference and maturation genes supports the reported hybrid character of subtype I-D CRISPR-Cas systems. We report tight and very high 5mC methylation of the CRISPR1 repeat sequences. Nevertheless, cells lacking the 5mC methylation activity were unaffected in their CRISPR1-mediated interference response but the efficiency of conjugation was reduced by 50%. These results point to an unknown role of m5CGATCG DNA-methylation marks in conjugation and DNA transformation.

Dataset Information

CRISPR/Cas System and Factors Affecting Its Precision and Efficiency.

Publications

CRISPR/Cas System and Factors Affecting Its Precision and Efficiency.

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