Cas9 Target Finder and PAM Finder

Find Cas9 target sites in a sequence on both strands: the PAM (NGG, NG or NNGRRT), the adjacent protospacer, its GC content, the cut position and basic flags.

Result

Estimate

Target sites

With no flag: 10 of 10

Target sites
Strand	Position	Protospacer (5′ → 3′)	PAM	GC %	Cut after base	Flags
-	8–27	TGGATCCTGAAGCTTAACGT	CGG	45	10	none
+	18–37	TCAGGATCCATGAAGCTGTA	CGG	45	34	none
-	28–47	AAGCTGGCCGTACAGCTTCA	TGG	55	30	none
+	35–54	GTACGGCCAGCTTGACCGTA	CGG	60	51	none
+	42–61	CAGCTTGACCGTACGGATCT	TGG	55	58	none
-	44–63	CAAGATCCGTACGGTCAAGC	TGG	55	46	none
+	53–72	TACGGATCTTGGAAGCTTCC	AGG	50	69	none
-	53–72	GGAAGCTTCCAAGATCCGTA	CGG	50	55	none
+	62–81	TGGAAGCTTCCAGGCTAGCC	TGG	60	78	none
-	74–93	TTCGAATATCCAGGCTAGCC	TGG	50	76	none

Note

This is simple sequence matching. It lists where the PAM and a full-length protospacer occur in the sequence you pasted; it does not predict cutting efficiency and does not search a genome, so it says nothing about off-target sites elsewhere. Check candidates with a genome-wide off-target tool, and validate experimentally.

Note

Flags: TTTT ends transcription from U6 and other Pol III promoters; GC outside 40 to 80% is commonly associated with poorer guides.

Significant figuresScientific notation

Calculation

Blunt cut 3 bp upstream of the PAM.
Sites are found by matching the PAM on both strands. Positions are 1-based on the sequence as entered; "cut after base" is on the top strand.

Formula

\text{SpCas9 PAM: NGG directly 3}'\text{ of a 20 nt protospacer}

\text{SaCas9 PAM: NNGRRT after a 21 nt protospacer}

\text{cut: 3 bp upstream of the PAM, blunt}

$\mathrm{PAM}$: protospacer adjacent motif, the short sequence the nuclease needs next to its target
$\text{protospacer}$: the target sequence matching the guide's spacer

How it works

Cas9 cuts only where its target is followed by a PAM. For the commonly used SpCas9 this is NGG, so a target site is any 20 bases lying immediately 5′ of an NGG. The tool finds every such site in the sequence you paste, on both strands, and reports where the nuclease would cut, three base pairs upstream of the PAM.

The engineered SpCas9-NG recognises a shorter NG PAM and so has far more sites, though it tends to act less efficiently at some of them. SaCas9 has a longer NNGRRT PAM and a slightly longer spacer. Each site is flagged for TTTT (a Pol III terminator) and for GC outside 40 to 80%, and for copies of the same protospacer within your sequence. Finding a site is the start of guide design, not the end.

Worked example

An illustrative 91 bp sequence, SpCas9 with a 20 nt spacer.

Every NGG on the top strand with 20 bases before it is a site, and every CCN on the top strand is an NGG site on the bottom strand.
The first site on the top strand is at 18 to 37, with PAM CGG; Cas9 cuts 3 bp upstream of the PAM, after base 34.

Ten target sites across the two strands, none flagged.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

Sequence matching only, to the PAM and the protospacer length you choose.
Sites are searched only in the sequence you paste, not across a genome.
No cutting efficiency or specificity is predicted.

Common mistakes

Treating a site with a matching PAM as a good guide. Efficiency varies enormously between sites.
Skipping a genome-wide off-target search, which is essential for guides used in cells or animals.
Forgetting that a target whose PAM is destroyed by the edit will not be re-cut, and one whose PAM remains will.

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Cas9 Target Finder and PAM Finder

Result

Calculation

Formula

How it works

Worked example

Assumptions

Common mistakes

Related tools

Result

Calculation