An easy and efficient method for gene knock-in
The PITCh (Precise Integration into Target Chromosome) systems are the alternative methods of CRISPR-Cas9- and TALEN-mediated knock-in
These systems enable seamless gene knock-in with extremely short homologous sequences (10–40 bp), which can be easily added to the donor vector by PCR and
In-Fusion cloning 1 .
The PITCh knock-in is based on one of the repair mechanism of DNA double strand break,microhomology-mediated end-joining (MMEJ). Since MMEJ is independent on homologous recombination (HR), which is mainly used for gene knock-in, PITCh system can be utilized in cells and organisms with low HR frequency.
The PITCh systems have currently been applied in cultured cells (HEK293T, HeLa, HCT116, CHO), silkworms, frogs, zebrafish, and mice.
PITCh designer 2.x is a free online tool to design
sgRNA and homology arms for gene knock-in.
This tool only needs sequence around the target region. After the sequence submission, followed by the selection of the target base, the application automatically designs sgRNA, microhomologies, and primers for constructing a donor vector for PITCh knock-in and for genotyping.
Go to the PITCh designer 2.x >>>
Paste the FASTA-formatted sequence around the target region, and click submit.
|H||A, C, T||not G|
|B||C, G, T||not A|
|V||A, C, G||not T|
|D||A, G, T||not C|
|N||A, C, G, T||aNy|
|Species||PAM sequence ( 5' -> 3' )|
|Streptococcus pyogenes (SP) ; SpCas9||NGG|
|Staphylococcus aureus (SA) ; SaCas9||NNGRRT or NNGRR(N)|
|Neisseria meningitidis (NM) ; NmCas9||NNNNGATT|
|Streptococcus thermophilus (ST) ; St1Cas9||NNAGAAW|
The application displays nucleotide buttons, which are color-coded according to their targetability and molecular species.
|Gray||Out of range||Any|
After selecting the desired position, the webtool begins to design the sequences for PITCh knock-in.
The design results consist of sequence information and graphical images of knock-in and donor vector construction ( as shown in the left figure ).Downloadable items
The PITCh designer 2.x can not only automatically select best primer pair for genotyping but help users to manually choose it on this viewer ( as shown in the right figure ).
This viewer can show primers for left junction / right junction / out-out PCR. By clicking on the arrow button on the left button form, you can change the primer type.
After clicking the left button for selecting primer pair, the viewer displays sequence information at the bottom of the graphical map. The sequence information can also be download by clicking the "DOWNLOAD SEQENCE" button.
The PITCh designer 2.1 can analyze the knock-in design and adds "efficiency annotations" to the knock-in design. We showed that the group of annotated knock-in design significantly achieved the high efficiency of precise knock-in compared with the no annotation group in HEK293T cells ( as shown in the right figure ).
The sequence features, which contribute to the gain of precise knock-in efficiency, will be shown in Desirable labels. On the other hand, Undesirable labels indicate the feature causing less knock-in efficiency.
The features were determined based on our previous research (Nakamae et al., 2022). The PITCh designer searches sequence context resulting in the mutation patterns affecting the knock-in efficiency.