PITCh KnockIn

An easy and efficient method for gene knock-in

What are the PITCh systems ?

 The PITCh (Precise Integration into Target Chromosome) systems are the alternative methods of CRISPR-Cas9- and TALEN-mediated knock-in 1 .


Advantages

 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.

Applications

 The PITCh systems have currently been applied in cultured cells (HEK293T, HeLa, HCT116, CHO), silkworms, frogs, zebrafish, and mice.







Web Application :
"PITCh designer"

PITCh designer 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.

Go to the PITCh designer >>>


Procedure

1. Paste sequence :


 Paste the FASTA-formatted sequence around the target region,
and click submit.

[NOTICE]
  • The acceptable length of the sequence is 100-15,000[bp].
  • The target base should not be located on either end of the sequence.
    The tool requires 48-bp flanking sequences on the both sides of the target base regardless of the lengths of microhomologies.
PITCh designer :

1. Paste sequence :

Options :

 You can use design options for the PITCh knock-in.



1 : Reading frame

 Three reading frames or 'No frame' can be selected (as shown in the right figure).

Options :
  • Frame 1 (default)
  • Frame 2
  • Frame 3
  • No frame
[NOTICE]
  • With selecting 'No frame', the design results are
    displayed with frame 1 without codon specification.
...

2 : Adjustment of reading frame

 PITCh designer offers two methods to prevent frameshifting.

Options :
  • 5'C-insertion (default)

    C-insertion mode fills in cytosine base(s) to adjust the reading frame.
    The number of fill-in bases depends on the selected frame and the position of knock-in.

  • Codon deletion

    Codon deletion mode removes extra base(s).
...

3 : Knock-in cassette

 The tool designs the primers for the construction of a targeting vector to knock-in the following two cassettes.

Options :
  • CMV-EGFP2APuroR-polyA

    Usage :
    • Transgenesis or knock-in/knock-out ( Nakamae et al., unpublished )

...

The plasmids for EGFP2APuroR knock-in are available from Addgene:
https://www.addgene.org/browse/article/16395/

4-6 : Microhomology lengths and PAM sequence requirement


4,6 : Lengths of left/right microhomologies
 The acceptable length of each microhomology arm is 10-40[bp]
(The default length is 20 bp.)

5 : PAM sequence requirement
 PAM sequence can be defined using IUB codes.
(The default sequence is 5'-NGG-3' ; SpCas9)

...
IUB codes 2
Code Definition Mnemonic
G G Guanine
A A Adenine
T T Thymine
C C Cytosine
R A, G puRines
Y C, T pYrimidines
M A, C aMino
K G, T Keto
S C, G Strong
W A, T Weak
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
PAM sequences 3
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

2. Select target :

 The application displays nucleotide buttons, which are color-coded according to their targetability and molecular species.

Color Targetability Base
Green Adenine
Red Thymine
Blue Guanine
Yellow Cytocine
Light Blue Any
Gray Out of range Any

 After selecting the desired position, the webtool begins to design the sequences for PITCh knock-in.

3. Show Result :

 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
  • Summary
    • The designed sequences are exported to tab-delimited text or CSV format, which can be viewed in Microsoft Excel.

  • Graphical image of knock-in
  • Graphical image of donor vector construction
2. Select target:

3. Show Result :