Engineering of Systems, Methods and Optimized Guidance Compositions for Sequence Manipulation
A technology of engineering and composition, applied in genetic engineering, biochemical equipment and methods, determination/inspection of microorganisms, etc.
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example 1
[0184] Example 1: CRISPR complex activity in the nucleus of eukaryotic cells
[0185] An exemplary type II CRISPR system is the type II CRISPR locus from S. pyogenes SF370, which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1 and two non-coding RNA elements tracrRNA and a short sequence composed of non-repetitive sequences. A characteristic array of repeated sequences (direct repeats) spaced apart by segments (spacers, about 30 bp each). In this system, targeted DNA double-strand breaks (DSBs) are generated in four sequential steps ( Figure 2A ). In the first step, two noncoding RNAs, pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. In the second step, the tracrRNA is hybridized to the direct repeat of the pre-crRNA, which is then processed into a mature crRNA containing a separate spacer sequence. In the third step, the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via ...
example 2
[0201] Example 2: CRISPR System Modifications and Alternatives
[0202] The ability to program sequence-specific DNA cleavage using RNA defines a new class of genome engineering tools for a variety of research and industrial applications. Several aspects of the CRISPR system can be further improved to increase the efficiency and versatility of CRISPR targeting. Optimal Cas9 activity can be dependent on free Mg present in mammalian nuclei 2+ High levels of free Mg 2+ Availability of NGG motifs (see e.g. Jinek et al., 2012, Science, 337:816), and a preference for NGG motifs located just downstream of protospacers limits targeting to the human genome average capacity per 12-bp. Some of these constraints can be overcome by exploring the diversity of CRISPR loci across microbial metagenomes (see eg Makarova et al., 2011, Nat Rev Microbiol, 9:467). Other CRISPR loci can be grafted into the mammalian cell environment by methods similar to those described in Example 1. showed tha...
example 3
[0203] Example 3: Sample target sequence selection algorithm
[0204] A software program is designed to identify candidate CRISPR target sequences on both strands of the input DNA sequence based on the desired guide sequence length and CRISPR motif sequence (PAM) for the specified CRISPR enzyme. For example, 5'-N can be obtained by searching both the input sequence and the reverse x -NGG-3' to identify the target site of Cas9 from Streptococcus pyogenes with the PAM sequence NGG. Likewise, a target site for Cas9 of S. pyogenes CRISPR1 with the PAM sequence NNAGAAW can be identified by searching for 5'-Nx-NNAGAAW-3' both on the input sequence and the reverse complement of the input sequence. Likewise, 5'-N can be searched both on the input sequence and the reverse x -NGGNG-3' to identify the target site of Cas9 of Streptococcus pyogenes CRISPR3 with the PAM sequence NGGNG. Can be fixed by program or specified by user N x The value "x" in , such as 20.
[0205] Since multip...
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